A method for constructing an animal model of neurotrophic keratitis and its application

Abstract

The present invention provides a method for constructing an animal model of neurotrophic keratitis and its application, and relates to the technical field of animal models. Cre Mice were co-housed with ROSA26i-DTR mice, and the offspring were genotyped. After screening for TRPV1-DTR double-heterozygous mice, these TRPV1-DTR mice were intraperitoneally injected with diphtheria toxin. Twenty-one days after treatment, a mouse model of neurotrophic keratitis was established. This invention, developed using widely used experimental mice, is the first genetic NK model. The model is simple, stable, and has a short lifespan. It aligns with clinical staging and meets clinical disease progression criteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and in particular relates to a method for constructing a neurotrophic keratitis animal model and application thereof. Background Art

[0002] Neurotrophic keratitis (NK) is a degenerative corneal disease caused by impaired function of the corneal nerves innervated by the trigeminal nerve. With an incidence of less than 1.6 / 10,000, it is relatively rare and often missed or misdiagnosed. Corneal nerve damage leads to damage to corneal epithelial and stromal cells, which in turn affects the secretion of neurotrophic factors (such as nerve growth factor (NGF)) by these target cells, further exacerbating corneal nerve pathology. Furthermore, corneal nerve damage and decreased NGF can also lead to decreased tear secretion, further aggravating corneal pathology, leading to a vicious cycle and ultimately progressing to neurotrophic keratitis. The most critical clinical feature of this disease is decreased or absent ocular surface perception, which can lead to missed and misdiagnoses in clinical practice.

[0003] Basic research and drug treatment of NK urgently need suitable animal models, but there is currently a lack of stable and efficient NK animal models. Currently, the most commonly used animal model for NK research is stereotactic electrocautery injury to the ophthalmic branch of the trigeminal nerve, which is mostly modeled in rats, with a model success rate of approximately 60%. Rats can only survive 3-6 days after surgery. Due to the small size of mice and the difficulty of surgical operation, the model is not widely used. The second method is subcutaneous injection of capsaicin into newborn mice. The phenotype begins to appear at 3 weeks, the modeling cycle is long, and there are large individual differences. The severity of the disease depends to a certain extent on the dose of capsaicin used. Therefore, there is an urgent need for a simple and stable NK animal model with a short construction cycle that can be adapted to clinical staging and meets the standards of clinical disease progression. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for constructing an animal model of neurotrophic keratitis. The present invention uses mice that are widely used in experiments to establish the model. The model is simple and stable, has a short cycle, can be in line with clinical staging, and meets the standards of clinical disease progression.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing an animal model of neurotrophic keratitis, comprising the following steps: Cre The mice were co-housed with ROSA26i-DTR mice, and TRPV1-DTR double heterozygous mice were screened out. The TRPV1-DTR double heterozygous mice were then injected with diphtheria toxin. After 21 days of injection treatment, a neurotrophic keratitis mouse model was obtained.

[0007] Preferably, the injection frequency of the diphtheria toxin is once a day for 5 consecutive days and then rest for 2 days.

[0008] More preferably, the injection dose of the diphtheria toxin is 200 ng per mouse per injection.

[0009] Preferably, the diphtheria toxin injection is performed after the TRPV1-DTR double heterozygous mouse weighs more than 20 g.

[0010] More preferably, the diphtheria toxin is injected intraperitoneally.

[0011] Preferably, the method of screening TRPV1-DTR double heterozygous mice is to perform genotyping on the mice.

[0012] More preferably, the genotype identification method comprises the following steps: separating offspring mice from their parents 4 weeks after birth and marking them; obtaining genomic DNA of the offspring mice; then performing a PCR amplification reaction and identifying the PCR products using 2% agarose gel electrophoresis.

[0013] The present invention also provides a neurotrophic keratitis animal model obtained by the above construction method.

[0014] The present invention also provides the use of the above-mentioned neurotrophic keratitis animal model in the preparation of a drug for treating neurotrophic keratitis.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] Compared with the control group, the animal model constructed by this invention showed significantly reduced corneal sensitivity and tear secretion in mice with TRPV1 sensory nerve ablation, and slit-lamp imaging revealed corneal epithelial defects. The corneal nerve density of mice with TRPV1 sensory nerve ablation was significantly reduced, and neovascularization increased significantly. OCT imaging and HE staining revealed significant corneal edema in the TRPV1 sensory nerve ablated mice, and corneal flat-mount staining also revealed a large infiltration of inflammatory cells. The ocular phenotype of TRPV1 sensory nerve ablated mice mimics the clinical classification of neurotrophic keratitis in patients, providing a highly effective and stable animal model for studying the pathogenesis and treatment of neurotrophic keratitis.

[0017] Clinical NK disease is divided into three stages: Stage I: decreased tear secretion and corneal epithelial punctate defects; Stage II: corneal epithelial detachment and stromal edema; and Stage III: corneal ulceration, stromal dissolution, and even perforation. The modeling cycle of the present invention is 21 days, and within 21 days, nearly all TRPV1 sensory nerve ablation mice have achieved modeling. The animal model constructed in this invention utilizes widely used experimental mice and is the first genetic NK model. The model is simple, stable, and has a short lifespan, aligning with clinical staging and meeting clinical disease progression criteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 TRPV1-DTR mice were identified, Figure 1 A is the result of genotyping of TRPV1-DTR mice. Figure 1 B is the immunofluorescence staining result of frozen sections of TRPV1-DTR mice;

[0019] Figure 2 The corneal phenotype of TRPV1 sensory nerve ablation mice is shown in Figure 2. Figure 2 A shows the changes in tear secretion at different time points during the DT injection process in TRPV1-DTR mice. Figure 2 B shows the changes in corneal sensitivity of TRPV1-DTR mice at different time points during DT injection. Figure 2 C is a macroscopic photo of the cornea of ​​TRPV1-DTR mice at different time points during DT injection; d, day; ns, not significant; ***P < 0.001;

[0020] Figure 3 The results of neurodegeneration and vascular ingrowth in TRPV1 sensory nerve ablation mice are shown in Figure 2. Figure 3 A is a gross photograph of the cornea of ​​TRPV1 sensory nerve ablation mice and control mice (left column) and corneal flat mount immunofluorescence staining (right column, β3-tubulin, green, indicates corneal nerves); Figure 3 B is the gross photograph of the cornea of ​​TRPV1 sensory nerve ablation mice and control mice (left column) and corneal flat mount immunofluorescence staining (right column, CD31, red, indicating corneal blood vessels); Figure 3 C is the qPCR detection of angiogenic factors in the corneas of TRPV1 sensory nerve ablation mice and control mice Vegfa Expression; ****P<0.0001;

[0021] Figure 4 The results showed that TRPV1 nerve ablation mice had enhanced corneal inflammation, Figure 4 A is the corneal OCT imaging of TRPV1 sensory nerve ablation mice and control mice (left column), HE staining of sections (middle column), and immunofluorescence staining of corneal flat mounts (right column, CD45, green, indicates corneal immune cells); Figure 4 B is the qPCR detection of inflammatory factors in the corneas of TRPV1 sensory nerve ablation mice and control mice Tnf-α, Il-6, Il-1β ****P<0.0001. DETAILED DESCRIPTION

[0022] The present invention provides a method for constructing an animal model of neurotrophic keratitis, comprising the following steps: CreMice were co-housed with ROSA26i-DTR mice, and TRPV1-DTR double heterozygous mice were screened out. The TRPV1-DTR double heterozygous mice were then injected with diphtheria toxin. After 21 days of injection, a neurotrophic keratitis mouse model was obtained. Cre Mice and ROSA26i-DTR mice were purchased from The Jackson Laboratory, and TRPV1 Cre The mouse catalog number is Jax: 017769; the ROSA26i-DTR mouse catalog number is Jax: 007900.

[0023] In the present invention, the injection frequency of the diphtheria toxin is once a day, for 5 consecutive days and then rest for 2 days.

[0024] In the present invention, the injection dose of diphtheria toxin is 200 ng per mouse per injection. In a specific embodiment of the present invention, 200 ng of diphtheria toxin (DT) is dissolved in 100 μL of PBS and then injected.

[0025] In the present invention, diphtheria toxin injection is performed after the TRPV1-DTR double heterozygous mice weigh more than 20 g. In a specific embodiment of the present invention, diphtheria toxin injection can be performed after the TRPV1-DTR double heterozygous mice weigh more than 20 g, that is, when the TRPV1-DTR double heterozygous mice are 8 to 12 weeks old.

[0026] In the present invention, the diphtheria toxin is injected into the peritoneal cavity.

[0027] In the present invention, the method for screening TRPV1-DTR double heterozygous mice is to perform genotyping on the mice.

[0028] In the present invention, the genotyping method includes the following steps: separating offspring mice from their parents four weeks after birth and marking them. Simultaneously, the tail tissue is removed. 200 μL of solution A (0.05 M NaOH) is added to the tail and incubated in a 95°C water bath for 40 minutes. 20 μL of solution B (1 M Tris (121.14)-10 mM EDTA (292.248)) is then added. The mixture is mixed and centrifuged, and the supernatant is collected to obtain mouse genomic DNA. PCR amplification is then performed, and the PCR products are identified using 2% agarose gel electrophoresis.

[0029] In the present invention, the PCR amplification conditions and primers are referred to the following website:

[0030] TRPV1 CreDetailed identification information for mice: https: / / www.jax.org / Protocol?stockNumber=017769&protocolID=35042; detailed identification information for ROSA26i-DTR mice: https: / / www.jax.org / Protocol?stockNumber=007900&protocolID=22285.

[0031] After PCR using the specific primers provided by the official website, genotype identification was performed based on the size of the amplified fragment, among which TRPV1 cre The amplified fragment of the mutant (Mut) mouse is about 475bp, and the amplified fragment of the wild type (WT) is 496bp; the amplified fragment of the mutant (Mut) mouse is 300bp, and the amplified fragment of the wild type (WT) is 603bp. Cre The offspring of mice co-housed with ROSA26i-DTR mice acquired TRPV1 cre The 475bp and 496bp fragments of ROSA26i-DTR were obtained from mice, and the 300bp and 603bp fragments of ROSA26i-DTR were obtained from mice.

[0032] The present invention also provides a neurotrophic keratitis animal model obtained by the above construction method.

[0033] The present invention also provides the use of the above-mentioned neurotrophic keratitis animal model in the preparation of a drug for treating neurotrophic keratitis.

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. Example 1

[0035] Select TRPV1 that is sexually mature and healthy (8 weeks to 6 months old) CreMice were housed with ROSA26i-DTR mice in a 1:1 ratio of male to female. Offspring mice were born about 21 days later, and the genotype of the offspring mice was identified. The offspring mice were separated from their parents 4 weeks after birth, and the tails of the mice were marked with a line with a marker to distinguish each mouse. The tail tissue was cut, 200 μL of solution A (0.05M NaOH) was added to the tail and placed in a 95°C water bath for 40 minutes, 20 μL of solution B (1M Tris-10mMEDTA) was added, mixed and centrifuged, and the supernatant was obtained to obtain mouse genomic DNA. PCR amplification reaction was then performed, and the PCR products were identified using 2% agarose gel electrophoresis to screen out TRPV1-DTR double heterozygous mice, namely TRPV1-DTR mice (such as Figure 1 A). According to Figure 1 A shows that TRPV1 cre The amplified fragment of the mutant (Mut) mouse is approximately 475bp, and the amplified fragment of the wild type (WT) is 496bp; the amplified fragment of the mutant (Mut) mouse is 300bp, and the amplified fragment of the wild type (WT) is 603bp. The blank control is used as a negative control, and no amplified fragment is detected. TRPV1-DTR double heterozygous mice are TRPV1 Cre The offspring of mice co-housed with ROSA26i-DTR mice acquired TRPV1 cre The 475bp and 496bp fragments of ROSA26i-DTR were obtained from mice, and the 300bp and 603bp fragments of ROSA26i-DTR were obtained from mice.

[0036] When TRPV1-DTR mice weighing more than 20 g (8-12 weeks old, both males and females) were injected intraperitoneally with diphtheria toxin, once a day, with each mouse injected with 200 ng of DT (dissolved in 100 μL PBS), for 5 consecutive days and then stopped for 2 days, and this cycle was repeated until 21 days. In the control group, TRPV1-DTR mice were intraperitoneally injected with the same volume of solvent, i.e., 100 μL PBS. TRPV1 was immunofluorescently stained on frozen sections of mouse trigeminal ganglia (1:100, Santa Cruz, sc398417). According to Figure 1 As can be seen in B, compared with the control group TRPV1-DTR+PBS, the number of TRPV1-positive (red) neurons in the trigeminal ganglion of TRPV1-DTR+DT mice was significantly reduced, indicating the ablation of TRPV1-positive neurons in the trigeminal ganglion of TRPV1-DTR+DT mice.

[0037] 1. The ocular phenotype of mice with Trpv1 sensory nerve ablation was assessed by measuring corneal sensitivity (Luneau Ophtalmologie, Chartres Cedex, France), tear secretion (#30059010, Jingming, Tianjin, China), and slit lamp (TOPCON) imaging. Decreased corneal sensitivity, decreased tear secretion, and corneal epithelial defects indicate a NK stage I phenotype in the mouse cornea. The following steps were performed:

[0038] Tear production was measured using a phenol red cotton thread (#30059010, Jingming, Tianjin, China). The thread was placed on the outer third of the palpebral conjunctiva for 20 seconds. The thread turned red after being soaked with tears, and the length (mm) of the thread represents tear production. (Normal tear production in mice is 6 mm.)

[0039] Corneal sensitivity was measured using a sensorimeter (Luneau Ophtalmologie, Chartres Cedex, France). All mice were unanesthetized. Measurements were made starting at the maximum length of the needle filament and shortened by 0.5 cm after three touches without a response. The corneal sensitivity threshold was the maximum length of the needle filament at which a positive response occurred. The measurements were recorded and repeated three times. (Normal corneal sensitivity in mice: 6 cm.)

[0040] according to Figure 2 The results showed that the tear secretion of TRPV1-DTR mice was significantly reduced on the third day after DT injection (e.g. Figure 2 A), corneal sensitivity begins to decrease (e.g. Figure 2 B). TRPV1-DTR mice exhibit corneal epithelial lesions that progress over time. On days 3-6 after DT injection, the cornea exhibits punctate epithelial defects, suggesting a NK phase I phenotype. By day 12, epithelial ulcers gradually develop in the mouse cornea, suggesting a NK phase II phenotype. Finally, corneal perforation develops on day 21, suggesting a NK phase III phenotype (e.g., Figure 2 C).

[0041] 2. Evaluate corneal neurodegeneration and vascular ingrowth in TRPV1-DTR mice by immunofluorescence staining of corneal flat mounts for the nerve fiber marker β3-tubulin (1:300, Biolegend, 657404) and the vascular marker CD31 (1:100, Biolegend, 102407). This involves the following steps:

[0042] Immunofluorescence staining of corneal flat mounts or sections: Mouse eyeballs were fixed with 4% paraformaldehyde, and the corneas were excised for flat mount staining. Eyeballs were frozen, embedded, and sectioned into 7-μm sections for section staining. All sections were then permeabilized, chamber-blocked, and incubated with primary antibodies overnight at 4°C. The next day, the sections were incubated with fluorescent secondary antibodies for 2 hours at room temperature. Images were obtained using a confocal fluorescence microscope (ZEISS) after staining with DAPI (Solarbio, Beijing, China).

[0043] according to Figure 3 The results showed that the corneas of TRPV1 sensory nerve ablation mice and control mice were stained for β3-tubulin and CD31. The corneal nerve density of DT-injected mice was significantly reduced, and the nerve fibers almost completely disappeared (e.g. Figure 3 A), New blood vessels invade the cornea (e.g. Figure 3 B), angiogenic factor Vegfa The expression of DT in the cornea of ​​mice was significantly upregulated (e.g. Figure 3 C).

[0044] 3. The present invention uses optical coherence tomography (OCT) imaging of the mouse cornea and hematoxylin and eosin (HE) (G1005, Servicebio, Wuhan, China) staining of mouse eyeball sections to evaluate corneal edema, and uses inflammatory factors ( Tnf-α 、 Il-6 、 Il-1β ) qPCR and immunofluorescence staining for the immune cell marker CD45 were used to assess corneal inflammation, including the following steps:

[0045] qPCR: Mouse corneal tissue was excised and thoroughly ground. Total RNA from corneal cells was extracted using the Nucleospin RNA Kit (Transgen, Beijing, China). Reverse transcription was then performed using the HiScript III RT SuperMix Kit (Vazyme, Nanjing, China). Real-time quantitative PCR was performed using SYBR Green reagent (Vazyme, Nanjing, China) and a Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

[0046] according to Figure 4 The results showed that the corneal thickness of TRPV1 sensory nerve ablation mice increased in OCT imaging, and there was an inflammatory response (such as Figure 4 A). HE staining showed corneal epithelial detachment and stromal edema and thickening in TRPV1 sensory nerve ablation mice (Fig. Figure 4A). Immunofluorescence staining of corneal flat mounts showed that the cornea of ​​TRPV1 sensory nerve ablation mice had a large number of inflammatory cells (CD45 positive) infiltrating (e.g. Figure 4 A). qPCR results showed that TRPV1 sensory nerve ablation in the cornea of ​​mice Tnf-α 、 Il-6 、 Il-1β Transcription levels were significantly upregulated (e.g. Figure 4 B).

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of neurotrophic keratitis, characterized in that: The steps include: Cre The mice were co-housed with ROSA26i-DTR mice, and TRPV1-DTR double heterozygous mice were screened out. The TRPV1-DTR double heterozygous mice were then injected with diphtheria toxin. After 21 days of injection treatment, a neurotrophic keratitis mouse model was obtained.

2. The construction method according to claim 1, characterized in that The injection frequency of the diphtheria toxin is once a day for 5 consecutive days and then rest for 2 days.

3. The construction method according to claim 2, characterized in that The injection dose of the diphtheria toxin was 200 ng per mouse per injection.

4. The construction method according to claim 1, wherein After the weight of TRPV1-DTR double heterozygous mice reached more than 20 g, diphtheria toxin injection was performed.

5. The construction method according to claim 4, characterized in that The diphtheria toxin is injected intraperitoneally.

6. The construction method according to claim 1, characterized in that The method for screening TRPV1-DTR double heterozygous mice is to perform genotyping on the mice.

7. The construction method according to claim 6, characterized in that The genotype identification method comprises the following steps: separating offspring mice from their parents after 4 weeks of birth and marking them; obtaining genomic DNA of the offspring mice; then performing PCR amplification reaction and identifying the PCR products using 2% agarose gel electrophoresis.

8. Use of the method for constructing an animal model of neurotrophic keratitis according to any one of claims 1 to 7 in the preparation of a drug for treating neurotrophic keratitis.

Images