Method for constructing an animal model for the study of ganglion cells and method for distinguishing between ganglion cell types

Abstract

The application provides a construction method of an animal model for ganglion cell research, comprising the following steps: mating a first target animal with a second target animal to obtain a hybrid first-generation animal, wherein the first target animal is photoreceptor degenerative, and the second target animal carries an LSL recognition site and a fluorescent protein gene; injecting a first virus suspension and a second virus suspension into the vitreous body of the hybrid first-generation animal to obtain an animal model, wherein the first virus suspension comprises a virus carrying a Cre recombinase gene, and the second virus suspension comprises a virus carrying a calcium indicator gene. The construction method is simple and convenient to operate, the obtained animal model is photoreceptor degenerative and has multiple markers, can be used for the differentiation of ganglion cell types, and is favorable for the selection of electrical stimulation parameters in a visual prosthesis, so that the visual prosthesis suitable for photoreceptor degeneration is obtained. The application further provides a differentiation method of ganglion cell types.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to methods for constructing animal models for ganglion cell research and methods for distinguishing ganglion cell types. Background Technology

[0002] Visual reconstruction via implantation of a visual prosthesis is a cutting-edge, clinically proven, and long-term effective solution for restoring basic vision. It utilizes microelectronic devices, employing implanted microelectrode arrays to stimulate ganglion cells in the visual pathway, thereby converting external image information into specific nerve impulses that are transmitted to the visual center. Ganglion cells can be categorized into ON and OFF types based on their response to light stimulation, reacting to increases and decreases in light intensity, respectively. If different types of ganglion cells can be selectively activated at the desired time or space, the visual prosthesis can elicit more physiologically accurate neural activity. Currently, the electrical stimulation methods in visual prostheses target normal ganglion cells. However, when retinal degeneration occurs, the photoreceptors at the front end of the visual pathway degenerate, rendering existing electrical stimulation methods inadequate. Therefore, distinguishing the types of ganglion cells with degenerated photoreceptors is a pressing issue that needs to be addressed in the application of visual prostheses. Summary of the Invention

[0003] In view of this, this application provides a method for constructing an animal model for ganglion cell research and a method for distinguishing ganglion cell types. The animal model has degenerated photoreceptors and has multiple markers that can be used to distinguish ganglion cell types, which is beneficial for the selection of electrical stimulation parameters in visual prostheses to obtain visual prostheses suitable for photoreceptor degeneration.

[0004] In a first aspect, this application provides a method for constructing an animal model for ganglion cell research, comprising:

[0005] The first target animal was mated with the second target animal to obtain a first-generation hybrid animal. The photoreceptors of the first target animal were degenerated, and the second target animal carried the LSL recognition site and fluorescent protein gene.

[0006] A first viral suspension and a second viral suspension were injected into the vitreous body of the first-generation heterozygous animal to obtain an animal model, wherein the first viral suspension included a virus carrying the Cre recombinase gene and the second viral suspension included a virus carrying the calcium indicator gene.

[0007] Optionally, the titer of the first virus suspension is greater than or equal to 5 × 10⁻⁶. 12 Gene copy number / ml, the titer of the second viral suspension is greater than or equal to 1×10⁻⁶. 13 Gene copy number / ml.

[0008] Optionally, the injection volume of the first virus suspension is 25 μl / kg-100 μl / kg, and the injection volume of the second virus suspension is 25 μl / kg-100 μl / kg.

[0009] Optionally, the viral vector carrying the Cre recombinase gene has a Nefh promoter.

[0010] Optionally, the fluorescent protein includes at least one of red fluorescent protein, blue fluorescent protein, and yellow fluorescent protein.

[0011] Furthermore, the red fluorescent protein includes at least one of tdTomato, DsRed, TagRFP, and mCherry.

[0012] Furthermore, the blue fluorescent protein includes at least one of EBFP, EBFP2, Azurite, mKalama1, and mTagBFP.

[0013] Furthermore, the yellow fluorescent protein includes at least one of EYFP, Topaz, Venus, mCitrine, and Ypet.

[0014] Optionally, the calcium indicator includes at least one of GCaMP1, GCaMP2, GCaMP3, GCaMP5, GCaMP6, GCaMP7, GCaMP-X, and RCaMPs.

[0015] Optionally, the first target animal and the second target animal are selected from at least one of mice, rats, rabbits, sheep and monkeys.

[0016] Optionally, the virus includes at least one of adenovirus, adeno-associated virus, lentivirus, and retrovirus.

[0017] Secondly, this application provides a method for distinguishing ganglion cell types, including:

[0018] The first target animal was mated with the second target animal to obtain a first-generation hybrid animal. The photoreceptors of the first target animal were degenerated, and the second target animal carried the LSL recognition site and fluorescent protein gene.

[0019] The first virus suspension and the second virus suspension were injected into the vitreous body of the first-generation heterozygous animal to obtain an animal model, wherein the first virus suspension included a virus carrying the Cre recombinase gene and the second virus suspension included a virus carrying the calcium indicator gene.

[0020] A retinal sample from the animal model was obtained, and the retinal sample was electrically stimulated and observed to obtain a first observation result.

[0021] The retinal sample was incubated with acetylcholine transferase antibody, and then observed after adding fluorescent dye-labeled anti-IgG antibody to obtain a second observation result;

[0022] The types of ganglion cells are distinguished based on the first and second observation results.

[0023] Optionally, the electrical stimulation parameters include a stimulation duration of 200ms-300ms, a stimulation frequency of 1kHz-6.25kHz, and a stimulation amplitude of 1μA-240μA.

[0024] Optionally, the fluorescent dye includes at least one of the following: ALEXA Fluor series dyes, fluorescein isothiocyanate, rhodamine, phycoerythrin, and anthocyanins.

[0025] This application provides a method for constructing an animal model for ganglion cell research. This method is simple and easy to operate. The resulting animal model has degenerated photoreceptors and multiple markers, which can be used to distinguish ganglion cell types. This application also provides a method for distinguishing ganglion cell types, which can differentiate the types of ganglion cells with degenerated photoreceptors. This is beneficial for the selection of electrical stimulation parameters in visual prostheses and helps to obtain visual prostheses suitable for degenerated photoreceptors. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. The specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0027] Figure 1 This is a flowchart illustrating a method for constructing an animal model for ganglion cell research according to an embodiment of this application.

[0028] Figure 2 A method for distinguishing ganglion cell types provided in one embodiment of this application.

[0029] Figure 3 A microscope image showing the results of one embodiment of this application.

[0030] Figure 4 A microscope image showing a result of another embodiment of this application.

[0031] Figure 5 A flowchart is provided for Example 1 showing a method for constructing an animal model for ganglion cell research.

[0032] Figure 6The waveform diagram is for the electrical stimulation used in Example 2. Detailed Implementation

[0033] The following are preferred embodiments of this application. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of this application, and these improvements and modifications are also considered to be within the scope of protection of this application.

[0034] Please see Figure 1 The above is a flowchart illustrating a method for constructing an animal model for ganglion cell research according to an embodiment of this application, including:

[0035] S101: The first target animal is mated with the second target animal to obtain a first-generation heterozygous animal. The photoreceptors of the first target animal are degenerated, and the second target animal carries the LSL recognition site and fluorescent protein gene.

[0036] S102: Inject the first virus suspension and the second virus suspension into the vitreous body of a hybrid generation animal to obtain an animal model, wherein the first virus suspension includes a virus carrying the Cre recombinase gene and the second virus suspension includes a virus carrying the calcium indicator gene.

[0037] Ganglion cells can be classified into ON-type and OFF-type cells based on their response patterns to light stimulation, reacting to increases and decreases in light intensity, respectively. Currently, the electrodes used in clinically implanted visual prostheses are too large in diameter, resulting in each electrode simultaneously stimulating dozens or even hundreds of ganglion cells. ON-type and OFF-type ganglion cells are often activated simultaneously during stimulation, significantly reducing the resolution of the visual prosthesis. Therefore, selectively activating different types of ganglion cells has become a critical bottleneck in the field of visual prostheses. For example, selective activation of ganglion cells can be achieved by optimizing the waveform of high-frequency electrical stimulation. However, when photoreceptors degenerate, the electrophysiological characteristics of ganglion cells change, rendering the above results meaningless. Furthermore, patch-clamp recording is currently commonly used to distinguish between ON-type and OFF-type ganglion cells. Once the electrophysiological characteristics of ganglion cells change, theoretically, it is no longer possible to distinguish them using electrical recording. Patch-clamp recording also requires cell membrane disruption, making the technique complex, difficult to operate, and time-consuming. Therefore, how to distinguish different types of retinal ganglion cells under photoreceptor degeneration, and thus further achieve selective stimulation, has become a key technical challenge. This application provides a method for constructing an animal model for ganglion cell research. By constructing an animal model with multiple markers for ganglion cell research, different types of ganglion cells can be distinguished, thereby achieving selective stimulation and improving the resolution and quality of visual prostheses.

[0038] In this application, the photoreceptors of the first target animal are degenerated, the second target animal carries an LSL recognition site and a fluorescent protein gene, and the resulting hybrid generation animals have degenerated photoreceptors and carry an LSL recognition site (loxP-stop-loxP) and a fluorescent protein gene. The fluorescent protein gene is located downstream of the LSL recognition site. Due to the presence of the LSL recognition site, the second target animal and the hybrid generation animals cannot normally express fluorescent protein. By injecting a virus carrying a Cre recombinase gene, Cre recombinase is expressed in the hybrid generation animals. The Cre recombinase recognizes the loxP site and undergoes recombination, expressing the fluorescent protein gene downstream of loxP, thereby producing fluorescent protein in the cells of the hybrid generation animals. During electrical stimulation, a brief peak in calcium ion concentration occurs when neurons fire. Calcium ion concentration can characterize neuronal activity. By injecting a virus carrying a calcium indicator gene, a calcium indicator is expressed in the hybrid generation animals, allowing the calcium ion concentration to be expressed through fluorescence intensity during neuronal firing, while simultaneously obtaining the location of the stimulated neurons.

[0039] In one embodiment of this application, a loxP-stop-loxP fragment is provided, and a fluorescent protein gene is linked to the downstream of the loxP-stop-loxP fragment (LSL-fluorescent protein gene) and then knocked into an animal to obtain a second target animal.

[0040] In one embodiment of this application, the fluorescent protein includes at least one selected from red fluorescent protein, blue fluorescent protein, and yellow fluorescent protein. By selecting the aforementioned fluorescent proteins, cells can be stained more effectively. In one embodiment, the red fluorescent protein includes at least one selected from tdTomato, DsRed, TagRFP, and mCherry. In another embodiment, the blue fluorescent protein includes at least one selected from EBFP, EBFP2, Azurite, mKalama1, and mTagBFP. In yet another embodiment, the yellow fluorescent protein includes at least one selected from EYFP, Topaz, Venus, mCitrine, and Ypet.

[0041] In one embodiment of this application, the first target animal and the second target animal are selected from at least one of mice, rats, rabbits, sheep, and monkeys. In one embodiment, the first target animal may be an rd1 mouse, and the second target animal may be an Ai14 mouse.

[0042] In one embodiment of this application, the virus includes at least one of adenovirus, adeno-associated virus (AAV), lentivirus, and retrovirus. In one embodiment, the virus is AAV.

[0043] In one embodiment of this application, the viral vector carrying the Cre recombinase gene has a Nefh promoter. The Nefh promoter is a ganglion cell-targeting promoter, thereby causing the production of Cre recombinase in ganglion cells, which in turn causes the expression of a fluorescent protein in the ganglion cells, thereby specifically labeling the ganglion cells. In one embodiment, the fluorescent protein is tdTomato. After injection of the first viral suspension, the ganglion cells express tdTomato and produce red fluorescence.

[0044] In one embodiment of this application, the titer of the first virus suspension is greater than or equal to 5 × 10⁻⁶. 12 Gene copy number / ml, thus ensuring Cre recombinase expression in the cells of the first-generation heterozygous animals. Furthermore, the titer of the first viral suspension is greater than or equal to 6 × 10⁻⁶. 12 Gene copy number / ml. Furthermore, the titer of the first viral suspension is greater than or equal to 8 × 10⁻⁶. 12 Gene copy number / ml.

[0045] In one embodiment of this application, the injection volume of the first virus suspension is 25 μl / kg to 100 μl / kg. It is understood that kg represents the body weight of the first-generation heterozygous animal. Further, the injection volume of the first virus suspension is 30 μl / kg to 70 μl / kg. Specifically, the injection volume of the first virus suspension can be, but is not limited to, 30 μl / kg, 40 μl / kg, 45 μl / kg, 50 μl / kg, or 60 μl / kg, etc.

[0046] In one embodiment of this application, the calcium indicator includes at least one of GCaMP1, GCaMP2, GCaMP3, GCaMP5, GCaMP6, GCaMP7, GCaMP-X, and RCaMPs. In one embodiment, the calcium indicator is GCaMP6. GCaMP6 has excellent sensitivity and can accurately reflect the location of the stimulated neuron, ensuring the accuracy of subsequent neuronal cell type differentiation. It is understood that both fluorescent proteins and calcium indicators can produce color under fluorescent light. To better differentiate ganglion cells, the fluorescent protein and the calcium indicator exhibit different colors under fluorescent light.

[0047] In one embodiment of this application, the titer of the second virus suspension is greater than or equal to 1 × 10⁻⁶. 13 Gene copy number / ml, thus ensuring expression of the calcium indicator in the cells of the first-generation heterozygous animals. Furthermore, the titer of the second viral suspension is greater than or equal to 2 × 10⁻⁶. 13 Gene copy number / ml. Furthermore, the titer of the second viral suspension is greater than or equal to 3 × 10⁻⁶. 13 Gene copy number / ml.

[0048] In one embodiment of this application, the injection volume of the second virus suspension is 25 μl / kg to 100 μl / kg. Further, the injection volume of the second virus suspension is 30 μl / kg to 70 μl / kg. Specifically, the injection volume of the second virus suspension can be, but is not limited to, 30 μl / kg, 40 μl / kg, 45 μl / kg, 50 μl / kg, or 60 μl / kg, etc.

[0049] This application provides a method for constructing an animal model for ganglion cell research. The method is simple and easy to operate. The resulting animal model has degenerated photoreceptors and multiple markers, which can be used to distinguish ganglion cell types.

[0050] Please see Figure 2 The method for distinguishing ganglion cell types provided in one embodiment of this application includes:

[0051] S201: The first target animal is mated with the second target animal to obtain a first-generation heterozygous animal. The photoreceptors of the first target animal are degenerated, while the second target animal carries the LSL recognition site and fluorescent protein gene.

[0052] S202: Inject the first virus suspension and the second virus suspension into the vitreous body of a hybrid generation animal to obtain an animal model, wherein the first virus suspension includes a virus carrying the Cre recombinase gene and the second virus suspension includes a virus carrying the calcium indicator gene.

[0053] S203: Obtain retinal samples from animal models, electrically stimulate the retinal samples and observe them to obtain the first observation results.

[0054] S204: Retinal samples were incubated with acetylcholine transferase antibody, and then observed after adding fluorescently labeled anti-IgG antibody to obtain the second observation result.

[0055] S205: Differentiate the types of ganglion cells based on the first and second observation results.

[0056] The retina is primarily composed of three layers of nerve cell bodies and two layers of synaptic retina. From the inside of the eyeball outwards, these layers are: ganglion cell layer, inner reticular layer, nuclear layer, outer reticular layer, and outer nuclear layer. The ganglion cell layer contains ganglion cells, while the nuclear layer contains bipolar cells. The processes of ganglion cells and bipolar cells form synapses that constitute the outer reticular layer. ON-type ganglion cell synapses are closer to the ganglion cell layer, while OFF-type ganglion cell synapses are closer to the nuclear layer. Therefore, the dendritic terminals of ON-type and OFF-type ganglion cells are also close to the ganglion cell layer and nuclear layer, respectively, located in sublayer b and sublayer a of the inner reticular layer. This application, based on the natural layering of the retinal structure, uses calcium ion imaging and immunofluorescence staining techniques to differentiate and determine the ganglion cell types in animals with degenerated photoreceptors. Differentiating ganglion cell types based on the first and second observation results is beneficial for selecting selective stimulation parameters for visual prostheses. Furthermore, this method is simple, easy to operate, and contributes to improving the resolution and quality of visual prostheses.

[0057] The process of constructing the animal model in this application is described above and will not be repeated here.

[0058] In one embodiment of this application, obtaining a retinal sample from an animal model includes euthanizing the animal model, removing the eyeball, opening the cornea to expose the lens, removing the lens, opening the sclera, and completely peeling the retina through the pigment epithelium. It is understood that the order of electrical stimulation and immunostaining is not limited; electrical stimulation can be performed first, followed by the addition of acetylcholine transferase antibody and fluorescently labeled anti-IgG antibody, or acetylcholine transferase antibody and fluorescently labeled anti-IgG antibody can be added first, followed by electrical stimulation.

[0059] In one embodiment of this application, a retinal sample is observed before electrical stimulation. In one embodiment, a two-photon laser scanning microscope system is used to observe the retinal sample. It is understood that since the cells already express fluorescent proteins, the excitation wavelength can be adjusted to obtain observation results for the retinal sample.

[0060] In one embodiment of this application, the parameters of the electrical stimulation include a stimulation duration of 200ms-300ms, a stimulation frequency of 1kHz-6.25kHz, and a stimulation amplitude of 1μA-240μA. Specifically, the stimulation duration can be, but is not limited to, 200ms, 220ms, 250ms, 260ms, 280ms, 300ms, etc.; the stimulation frequency can be, but is not limited to, 1kHz, 1.67kHz, 2kHz, 2.5kHz, 3.33kHz, 4.17kHz, 5kHz, 6.25kHz, etc.; and the stimulation amplitude can be, but is not limited to, 1μA, 5μA, 20μA, 50μA, 100μA, 120μA, 150μA, 200μA, 220μA, etc. In one embodiment, during the electrical stimulation process, a two-photon laser scanning microscope system is used to observe the retinal sample, adjusting it to the excitation wavelength corresponding to the calcium indicator for observation.

[0061] In this application, the retinal astrocytes are the only cholinergic cells in the retina and can specifically express acetylcholine transferase. Therefore, the retinal retinal retina is labeled with acetylcholine transferase antibody and fluorescent dye-labeled anti-IgG antibody to obtain the morphology and structure of the retinal retina.

[0062] In one embodiment of this application, a stock solution containing acetylcholine transferase antibody is provided. The stock solution is diluted and then used to incubate fixed cells. The dilution factor can be, but is not limited to, 10-100. In another embodiment of this application, a stock solution containing fluorescently labeled anti-IgG antibody is provided. The stock solution is diluted and then used to incubate fixed cells. The dilution factor can be, but is not limited to, 10-800.

[0063] In one embodiment of this application, the fluorescent dye includes at least one selected from the ALEXA Fluor series dyes, fluorescein isothiocyanate, rhodamine, phycoerythrin, and anthocyanins. Specifically, the ALEXA Fluor series dyes include ALEXA Fluor 647, ALEXA Fluor 594, ALEXA Fluor 532, ALEXA Fluor 555, etc. In this application, the fluorescent protein, calcium indicator, and fluorescent dye can all produce color under fluorescent light. To better distinguish ganglion cells, the fluorescent protein, calcium indicator, and fluorescent dye exhibit different colors under fluorescent light.

[0064] Please see Figure 3 The image shown is a microscopic result image provided in one embodiment of this application. The white areas represent regions expressing fluorescent proteins; in this embodiment, this shows the location of ganglion cells. The dark gray areas represent immunofluorescence staining regions; in this embodiment, this shows the morphology of the inner retinal retina. Please refer to [link to relevant documentation]. Figure 4The image shown is a microscopic result image provided in another embodiment of this application. The white area is the location where the calcium indicator is colored, and in this embodiment, it shows the location and morphology of the stimulated ganglion cells. The dark gray area is the immunofluorescence staining area, and in this embodiment, it shows the morphology of the inner retinal retina.

[0065] The method for distinguishing ganglion cell types provided in this application is based on cell morphology and differentiates the types of ganglion cells with degenerated photoreceptors. This facilitates the selection of electrical stimulation parameters in visual prostheses and helps to obtain visual prostheses suitable for degenerated photoreceptors. Cre recombinase enables the expression of fluorescent proteins, and calcium ion imaging technology is used during electrical stimulation to show the conduction pathway of electrical signals in ganglion cells with degenerated / damaged photoreceptors. Combined with optical tools such as two-photon confocal microscopy, the resolution reaches the cellular level.

[0066] In this application, by distinguishing the types of ganglion cells, a closed-loop algorithm (such as Tianruo G, Yu YC, David T, et al. Closed-Loop Efficient Searching of Optimal Electrical Stimulation Parameters for Preferential Excitation of Retinal Ganglion Cells[J]. Frontiers in Neuroence, 2018, 12: 168-.) can be used to obtain the optimal stimulation parameters for stimulating ON-type or OFF-type cells, thereby enabling selective stimulation of ganglion cells and improving the resolution and quality of visual prostheses. In one implementation, bidirectional high-frequency electrical stimulation with a duration of 300ms was used. The stimulation frequencies were set to 1kHz, 1.67kHz, 2kHz, 2.5kHz, 3.33kHz, 4.17kHz, 5kHz, and 6.25kHz, with a stimulation amplitude range of 10μA-240μA. Each stimulation increment was 10μA, and a sufficient interval was required between stimulations to allow for cell recovery. The duration of both the cathode and anode phases was 40μs, and there was no time interval between the bidirectional currents. This method was used to stimulate ganglion cells and obtain the optimal stimulation parameters for different types of ganglion cells. This allowed for the acquisition of electrical stimulation parameters for the visual prosthesis, which is beneficial for improving the resolution and quality of the visual prosthesis.

[0067] Example 1: Method for constructing an animal model for ganglion cell research

[0068] The Ai14-rd1 mouse was obtained by mating the rd1 mouse with photoreceptor degeneration with the Ai14 mouse carrying the LSL recognition site and the tdTomato gene (LSL-tdTomato).

[0069] A 2251 bp Nefh sequence (NM_010904.3) was amplified from mouse genomic DNA, and used to replace the CMV promoter in pAAV.CMV-Cre to obtain a virus carrying the Cre recombinase gene (AAV.Nefh-Cre). 5-6 week old Ai14-rd1 mice were selected, and 1 μL of the first virus suspension (5 × 10⁻⁶ mcg) was injected into the vitreous humor of their left eye. 12 (Gene copy number / ml), the first viral suspension contains a virus carrying the Cre recombinase gene, and tdTomato is expressed after about 96 hours.

[0070] 1 μL of the second virus suspension (1×10⁻⁶) was injected into the vitreous body of his left eye. 13 Gene copy number / ml), the second viral suspension includes viruses carrying the calcium indicator gene (rAAV-hSyn-axon-GCaMP6s), to obtain an animal model; please refer to Figure 5 The flowchart below provides a method for constructing an animal model for ganglion cell research, as described in Example 1.

[0071] Example 2: Method for Differentiating Ganglion Cell Types

[0072] After anesthetizing the animal model obtained in Example 1 with an intraperitoneal injection of 50 mg / kg ketamine, the eyeball was quickly removed and the animal was euthanized by cervical dislocation. A small incision was made at the corneal rim, and the eyeball was placed in Ames culture medium pre-filled with 95% O2 and 5% CO2 for 30 minutes. The cornea was opened with microforceps to expose the lens, which was then carefully removed. The sclera was opened, and the retina was completely dissected through the pigment epithelium.

[0073] Retinal samples were rapidly transferred to a microscope stage equipped with a water immersion objective and a Nikon fluorescence illumination system. The retinal samples were continuously perfused (6 ml / min) with O2-saturated Ames solution in a NaHCO3-CO2 buffer system, and the perfusion solution was heated to a temperature of 31-33°C. Under the water immersion objective, observation was performed in MP-HDD mode using the two-photon microscope control software. A MaiTai laser was used, with the excitation wavelength set to 554 nm. The ganglion cell region was located by moving the stage. A resolution of 512×512 was selected, and the morphology of ganglion cells expressing red fluorescence was recorded by photographing.

[0074] A 25 μm diameter platinum-iridium stimulation electrode was selected and placed 40 μm from the retinal sample using a micromanipulator. Current was supplied to the microelectrode using a stimulator. The excitation wavelength was set to 920 nm, employing a cathode-priority, charge-balanced bidirectional current pulse stimulation. The stimulation duration was 300 ms, with stimulation frequencies of 1 kHz, 1.67 kHz, 2 kHz, 2.5 kHz, 3.33 kHz, 4.17 kHz, 5 kHz, and 6.25 kHz. The stimulation amplitude range was 10 μA–240 μA, incrementing by 10 μA each time, with sufficient interval between stimulations to allow for cell recovery. The pulse width (phase width) for both the cathode and anodic phases was 40 μs, with no time interval between bidirectional currents. The stimulation waveform is shown below. Figure 6 As shown. Photographs were taken in the microscope's T-stack mode, with a frame rate of 5fps, to obtain the electrical stimulation results.

[0075] Retinal samples were fixed with 4% paraformaldehyde fixative for 1.5 h, then eluted 6 times with phosphate buffer (0.5% Triton X-100, 0.1% NaN3, pH 7.4) for 30 min each time, followed by blocking with phosphate buffer (3% donkey serum) for 2 days. Acetylcholinesterase antibody was added and incubated for 5-7 days. After elution, the samples were incubated at 4°C for 2 days with ALEXA Fluor 647-labeled donkey anti-sheep IgG.

[0076] Under a two-photon microscope, images were taken again at a wavelength of 450 nm to obtain immunofluorescence detection results. The morphology and location of all ganglion cells could be determined based on the expressed red fluorescence. The location of the stimulated ganglion cells could be determined through electrical stimulation results. The layered morphology of the inner retinal retina was obtained through immunofluorescence detection. Based on whether the stimulated ganglion cell synapse was closer to the ganglion cell layer or the nuclear lamina, it could be determined whether the electrically stimulated cell was an ON-type or OFF-type cell.

[0077] This application utilizes an animal model to record retinal ganglion cell signal transduction through calcium ion fluorescence imaging, immunostaining imaging, and electrical stimulation two-photon image acquisition. This technique avoids patch-clamp recording, maintains cell integrity, reduces operational difficulty, and avoids artifacts in electrical recording. Furthermore, it can differentiate between different types of ganglion cells with degenerated photoreceptors, facilitating the optimization of selective stimulation parameters for visual prostheses and improving their resolution and biomimetic performance.

[0078] The above provides a detailed description of the embodiments provided in this application. This document elucidates and explains the principles and implementation methods of this application. The above description is only intended to help understand the methods and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for constructing an animal model for ganglion cell research, characterized in that, include: The first target animal was mated with the second target animal to obtain a first-generation hybrid animal. The first target animal was an rd1 mouse with photoreceptor degeneration, and the second target animal was an Ai14 mouse carrying the LSL recognition site and the tdTomato fluorescent protein gene. The first and second viral suspensions were injected into the vitreous bodies of the first-generation heterozygous animals to obtain an animal model. The first viral suspension included an AAV virus carrying a Cre recombinase gene initiated by the Nefh promoter, and the second viral suspension included an AAV virus carrying a calcium indicator gene. The calcium indicator was at least one of GCaMP1, GCaMP2, GCaMP3, GCaMP5, GCaMP6, GCaMP7, and GCaMP-X.

2. The construction method as described in claim 1, characterized in that, The titer of the first virus suspension is greater than or equal to 5 × 10⁻⁶. 12 Gene copy number / ml, the titer of the second viral suspension is greater than or equal to 1×10⁻⁶. 13 Gene copy number / ml.

3. The construction method as described in claim 1, characterized in that, The injection volume of the first virus suspension is 25 μl / kg-100 μl / kg, and the injection volume of the second virus suspension is 25 μl / kg-100 μl / kg.

4. A method for distinguishing ganglion cell types, characterized in that, include: The first target animal was mated with the second target animal to obtain a first-generation hybrid animal. The first target animal was an rd1 mouse with photoreceptor degeneration, and the second target animal was an Ai14 mouse carrying the LSL recognition site and the tdTomato fluorescent protein gene. The first and second viral suspensions were injected into the vitreous bodies of the first-generation heterozygous animals to obtain an animal model. The first viral suspension included an AAV virus carrying a Cre recombinase gene initiated by the Nefh promoter, and the second viral suspension included an AAV virus carrying a calcium indicator gene. The calcium indicator was at least one of GCaMP1, GCaMP2, GCaMP3, GCaMP5, GCaMP6, GCaMP7, and GCaMP-X. A retinal sample from the animal model was obtained, and the retinal sample was electrically stimulated and observed under a two-photon microscope to obtain a first observation result. The parameters of the electrical stimulation included a stimulation duration of 200ms-300ms, a stimulation frequency of 1kHz-6.25kHz, and a stimulation amplitude of 1μA-240μA. The retinal sample was incubated with acetylcholine transferase antibody, and then anti-IgG antibody labeled with ALEXA Fluor647 fluorescent dye was added. The sample was then observed using a two-photon microscope to obtain a second observation result. Based on the first observation result, the location of the stimulated ganglion cells is determined. Based on the second observation result, the layered morphology of the inner retinal network is obtained. Based on whether the stimulated ganglion cell synapse is closer to the ganglion cell layer or the nuclear layer, it is determined whether the electrically stimulated cell is an ON-type cell or an OFF-type cell.

Images