Method for constructing optic nerve injury model of Xenopus tropicalis, model and application method

Abstract

The present invention relates to a method for constructing an optic nerve injury model of a tropical clawed frog, a model and an application method thereof, wherein the modeling is implemented as follows: under microscopic observation, laser is used to damage the retrobulbar optic nerve of an anesthetized juvenile tropical clawed frog, thereby obtaining a tropical clawed frog optic nerve injury model; the method of using laser to damage the retrobulbar optic nerve of an anesthetized juvenile tropical clawed frog to obtain the tropical clawed frog optic nerve injury model, which is first of its kind in China and abroad, has the following advantages: the tropical clawed frog's body is transparent when young, and the laser can be directly observed, accurately focused, and quantitatively damaged; this optic nerve injury modeling method is accurate, controllable, has a high success rate, and has a low mortality rate; the tropical clawed frog's genome is highly homologous to the human genome, and its eyeball, retina, optic nerve, visual pathway tissue structure, and visual information conduction pathway are highly similar to those of humans; the tropical clawed frog's optic nerve is regenerative, which is conducive to the study of regeneration and repair mechanisms after injury, and opens up new approaches and methods for optic nerve protection research.

Description

Technical Field

[0001] The present invention relates to the technical field of optic nerve models, and more particularly to a method for constructing a tropical clawed frog optic nerve injury model, a tropical clawed frog optic nerve injury model and an application method thereof. Background Art

[0002] Optic nerve protection has always been a hot topic and a difficult problem in clinical and basic research. The establishment of animal models of optic nerve injury is very important for the development of optic nerve protection research. Currently, most animal models of optic nerve injury use rodents (such as rats and mice) or non-mammalian vertebrates (such as zebrafish), and direct or indirect optic nerve injury is caused by direct severance, compression, traction, or hydraulic shock.

[0003] The existing method has the following defects:

[0004] 1. The optic nerve is located at the back of the eyeball and deep in the eye socket, and is rich in blood vessels and muscle tissue around it. Direct damage to the optic nerve by clamping, cutting, pulling, etc. will cause damage to the tissues around the surgical approach, such as retrobulbar hemorrhage.

[0005] 2. Clamping the optic nerve under direct vision requires excessive traction on the eyeball, and the clamping position is difficult to be accurate and consistent. Optic nerve damage cannot be quantified, making it difficult to achieve consistency in optic nerve damage in model animals.

[0006] 3. Microsurgery is performed on animals under general anesthesia. The operator needs to be proficient in relevant techniques, and the animals have a certain mortality and blindness rate.

[0007] 4. Indirect optic nerve injury methods such as hydraulic shock and craniocerebral impact injury not only cause damage to tissues other than the optic nerve, but also make it difficult to ensure the consistency of optic nerve injury in model animals.

[0008] 5. Currently commonly used rodents (such as rats and mice) are not suitable for studying the self-regeneration and repair of optic nerve after injury.

[0009] 6. Non-mammalian vertebrates (such as zebrafish) cannot be subjected to water-exit intervention in vivo and cannot construct a precise optic nerve injury model.

[0010] There is a need for an animal optic nerve model and a modeling method that can overcome the above-mentioned defects. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a method for constructing a tropical clawed frog optic nerve injury model, a tropical clawed frog optic nerve injury model and an application method thereof, in response to the above-mentioned defects of the prior art.

[0012] The technical solution adopted by the present invention to solve its technical problem is:

[0013] A method for constructing a tropical clawed frog optic nerve injury model is constructed, and the implementation method is as follows:

[0014] Under microscope observation, the retrobulbar optic nerve of anesthetized juvenile tropical clawed frog was damaged by laser to obtain the tropical clawed frog optic nerve injury model.

[0015] The method for constructing a tropical clawed frog optic nerve injury model of the present invention, wherein the juvenile period is 47-54 years.

[0016] The method for constructing a tropical clawed frog optic nerve injury model of the present invention, wherein the tropical clawed frog uses a tropical clawed frog at stage 52-54.

[0017] In the method for constructing a tropical clawed frog optic nerve injury model of the present invention, the laser adopts a short-pulse neodymium-doped yttrium aluminum garnet laser.

[0018] The method for constructing the tropical clawed frog optic nerve injury model of the present invention comprises the following steps: using laser to injure the retrobulbar optic nerve of the anesthetized tropical clawed frog.

[0019] The laser was quantitatively fired N times at the retrobulbar optic nerve of anesthetized tropical clawed frogs.

[0020] The method for constructing a tropical clawed frog optic nerve injury model of the present invention comprises the following steps: the energy range of each laser emission is 0.4-0.8 mJ.

[0021] The method for constructing a tropical clawed frog optic nerve injury model of the present invention comprises the step of emitting laser energy of 0.6 mJ each time.

[0022] A tropical clawed frog optic nerve injury model is constructed by the tropical clawed frog optic nerve injury model construction method as described above.

[0023] A method for applying a tropical clawed frog optic nerve injury model, wherein the tropical clawed frog optic nerve injury model is applied in optic nerve protection research or treatment.

[0024] The beneficial effects of the present invention are: the use of laser to damage the retrobulbar optic nerve of anesthetized juvenile tropical clawed frog to obtain a tropical clawed frog optic nerve injury model, which is the first of its kind in China and abroad, has the following advantages: the body of the tropical clawed frog is transparent when young, and the laser can be directly viewed, accurately focused, and quantitatively damaged. This optic nerve injury modeling method is accurate, controllable, has a high success rate, and a low mortality rate; the genome of the tropical clawed frog has many similarities with the human genome, and the tissue structure of its eyeball, retina, and optic nerve is similar to that of the human eye; the tropical clawed frog optic nerve can regenerate, which is conducive to the study of regeneration and repair mechanisms after injury, and opens up new paths and methods for optic nerve protection research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the present invention will be further described below with reference to the accompanying drawings and embodiments. The drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive efforts.

[0026] Figure 1 This is the appearance of the tropical clawed frog's head, with the arrow indicating the right optic nerve;

[0027] Figure 2 This is a photo of the appearance of a juvenile tropical clawed frog, with the arrow indicating the left optic nerve;

[0028] Figure 3 This is a screenshot from a video of the tropical clawed frog laser modeling process. The red part indicates the laser focus.

[0029] Figure 4 This is a HE-stained view of a pathological section of the tropical clawed frog optic nerve after laser injury. DETAILED DESCRIPTION

[0030] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the following will be a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

[0031] Example 1

[0032] The method for constructing a tropical clawed frog optic nerve injury model according to a preferred embodiment of the present invention is implemented as follows:

[0033] Under microscope observation, the retrobulbar optic nerve of anesthetized juvenile tropical clawed frog was damaged by laser to obtain the tropical clawed frog optic nerve injury model.

[0034] As an amphibian, the tropical clawed frog can be used to model optic nerve damage in an out-of-water environment, with relatively simple procedures and a high success rate. The tropical clawed frog's genome is highly similar to the human genome, making it evolutionarily closer to mammals than zebrafish. The tropical clawed frog's simple breeding conditions and rapid development allow us to microinject and manipulate a large number of embryos at the same developmental stage, facilitating the construction of transgenic models and simultaneously collecting extensive embryonic biological information, conducting genetic screening, and conducting comparative studies.

[0035] It should be noted that the childhood period mentioned above is from 47 to 54 years;

[0036] Preferably, the tropical clawed frog is a 52-54 stage tropical clawed frog. After the 52-54 stage tropical clawed frog model is completed and resuscitation is completed, the frog's activities and feeding are observed to be normal, and the mortality rate is low.

[0037] Preferably, the laser uses a short-pulse neodymium-doped yttrium aluminum garnet laser, and the microscope uses a slit lamp microscope. The short-pulse neodymium-doped yttrium aluminum garnet (Nd:YAG) laser is a laser with extremely short pulses, high power, and a small spot size. When it is focused on the optic nerve axon, it causes the target tissue in a small area at the focus to be ionized and transformed into plasma. The plasma rapidly expands, generating shock waves and acoustic (pressure) waves, mechanically splitting the tissue around the focus, thereby achieving the purpose of precisely damaging the optic nerve.

[0038] Of course, it is understandable that, in addition to the above-mentioned YAG laser method, other existing types of lasers that can achieve similar effects can also be used, and simple transformations based on this principle also fall within the scope of protection of this application;

[0039] The skin of tropical clawed frogs at stages 52-54 is transparent, and the entire course of the optic nerve from leaving the eyeball to entering the central nervous system can be observed under a slit lamp microscope. The optic nerve and surrounding blood vessels can be directly observed under a slit lamp microscope, and the YAG laser can be precisely focused on the optic nerve. The successful modeling standard is that the optic nerve twitches after laser treatment without causing tissue bleeding.

[0040] Preferably, the method of damaging the retrobulbar optic nerve of the anesthetized tropical clawed frog using laser is:

[0041] The retrobulbar optic nerve of anesthetized tropical clawed frogs was quantitatively shot with laser light N times; preferably, the laser energy range for each shot was 0.4-0.8 mJ; more preferably, the laser energy for each shot was 0.6 mJ; pathology confirmed that the optic nerve was damaged, the damage was significantly correlated with the laser energy and the number of shots, the modeling success rate was high, the repeatability and consistency were good, and the degree of damage could be quantitatively controlled; and preliminary observations showed that self-regeneration and repair changes had occurred 12 days after modeling.

[0042] The accompanying drawings are described as follows:

[0043] The tropical clawed frog is an amphibian model animal with a true diploid genetic background. The tropical clawed frog genome map shows that its genome has many similarities with the human genome. Its eyeball, retina and optic nerve tissue structure ( Figure 1 The arrow points to the optic nerve) is similar to the human eye;

[0044] The skin of tropical clawed frogs (stages 47-54) is transparent. Although there is a small amount of pigment on the skin, the tissues and organs in the frog's body can still be observed through the skin. The optic nerve can be directly observed through the skin under a microscope, providing the necessary conditions for laser focused injury ( Figure 2 The middle arrow points to the optic nerve. Figure 3 The middle arrow points to the laser spot. Meanwhile, during this period, clawed frogs can tolerate anesthesia and laser manipulation, and the mortality rate after modeling is low.

[0045] The tropical clawed frog has rapid embryonic development, in vitro fertilization, and a high reproductive output. It also has the potential for lifelong optic nerve regeneration, and its individual growth and maturation rates can be regulated by varying temperatures. This will facilitate subsequent research on optic nerve regeneration mechanisms and optic nerve protection interventions.

[0046] After quantitative laser optic nerve injury, pathology confirmed that the optic nerve was damaged accurately and the damage was significantly correlated with the laser energy and the number of shots. The modeling success rate was high, the repeatability and consistency were good, and the degree of damage could be quantitatively controlled. It was also preliminarily observed that self-regeneration and repair changes had occurred one week after modeling ( Figure 4 HE staining of the pathological sections showed damage to the optic nerve sheath above, and coexistence of tissue damage and repair.

[0047] Example 2

[0048] A tropical clawed frog optic nerve injury model, wherein the tropical clawed frog optic nerve injury model is constructed by the tropical clawed frog optic nerve injury model construction method as described above;

[0049] This optic nerve injury model has a precisely controllable amount of damage and does not cause damage to other tissues besides the optic nerve axons. It will also facilitate subsequent research on optic nerve regeneration mechanisms, optic nerve protection interventions, and other areas.

[0050] Example 3

[0051] A method for applying a tropical clawed frog optic nerve injury model, wherein the tropical clawed frog optic nerve injury model is applied in optic nerve protection research or treatment.

[0052] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such improvements and changes should fall within the scope of protection of the appended claims of the present invention.

[0053] Related information:

[0054] [1]KUMARAN AM,SUNDAR G,CHYE L T.Traumatic optic neuropathy: a review[J].Craniomaxillofac Trauma Reconstr,2015,8(1):31-41.

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[0056] [3]BURKE E G,CANSLER S M,EVANSON N K.Indirect traumatic opticneuropathy:modeling optic nerve injury in the context of closed head trauma[J].Neural Regen Res,2019,14(4):593-594.

[0057] [4]RUIZ-EDERRA J,GARCíA M,HERNáNDEZ M,et al.The pig eye as a novelmodel of glaucoma[J].Exp Eye Res,2005,81(5):561-569.

[0058] [5]BORODINSKY L N.Xenopus laevis as a Model Organism for the Study ofSpinal Cord Formation,Development,Function and Regeneration[J].Front NeuralCircuits,2017,11:90.

[0059] [6]AMAYA E,OFFIELD M F,GRAINGER R M.Frog genetics:Xenopus tropicalisjumps into the future[J].Trends Genet,1998,14(7):253-255.

[0060] [7]NAKAJIMA K,NAKAJIMA T,TAKASE M,et al.Generation of albino Xenopustropicalis using zinc-finger nucleases[J].Dev Growth Differ,2012,54(9):777-784.

[0061] [8]LEVKOVITCH-VERBIN H,QUIGLEY H A,MARTIN K R,et al.A model to studydifferences between primary and secondary degeneration of retinal ganglioncells in rats by partial optic nerve transection[J].Invest Ophthalmol VisSci,2003,44(8):3388-3393.

[0062] [9]LEVKOVITCH-VERBIN H,QUIGLEY H A,KERRIGAN-BAUMRIND L A,et al.Opticnerve transection in monkeys may result in secondary degeneration of retinalganglion cells[J].Invest Ophthalmol Vis Sci,2001,42(5):975-982.

[0063]

[10] Cindy X.Kha,Philip H.Son,Julia Lauper,et al.A model forinvestigating developmental eye repair in Xenopus laevis.Experimental EyeResearch 2018,169:38–47。

Claims

1. A tropical clawed frog optic nerve injury model, characterized in that: The method for establishing the tropical clawed frog optic nerve injury model is as follows: Under microscope observation, laser is used to cause damage to the retrobulbar optic nerve of anesthetized juvenile tropical clawed frog, thereby obtaining a tropical clawed frog optic nerve damage model; the laser is a short-pulse neodymium-doped yttrium aluminum garnet laser; the tropical clawed frog is a 52-54 stage tropical clawed frog; the method for causing damage to the retrobulbar optic nerve of the anesthetized tropical clawed frog using laser is: the laser is quantitatively emitted to the retrobulbar optic nerve of the anesthetized tropical clawed frog.

2. The tropical clawed frog optic nerve injury model according to claim 1, characterized in that The laser energy per shot ranges from 0.4 to 0.8 mJ.

3. The tropical clawed frog optic nerve injury model according to claim 2, characterized in that: The laser energy per shot is 0.6 mJ.

4. A tropical clawed frog optic nerve injury model application method, characterized in that: Use of the tropical clawed frog optic nerve injury model according to any one of claims 1 to 3 in optic nerve protection research or treatment.

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