Method for constructing and evaluating porcine retinitis pigmentosa disease model

Abstract

The invention provides a construction and evaluation method of a porcine retinitis pigmentosa disease model, and belongs to the technical field of animal models of human diseases. The invention provides a method for constructing and evaluating a porcine retinitis pigmentosa disease model, which comprises the following steps: applying iodoacetic acid to the vein of a miniature pig to construct the porcine retinitis pigmentosa disease model; after the iodoacetic acid is applied to the veins of the miniature pig, the miniature pig is subjected to ophthalmic examination; the ophthalmic examination comprises eye morphological structure and function examination. According to the construction method, iodoacetic acid is used as a modeling reagent, directional damage to the miniature pig retina photoreceptor cells can be rapidly achieved within one week, and the modeling rate is high. Moreover, after the iodoacetic acid is applied to the veins of the miniature pig, the miniature pig is subjected to ophthalmic examination including eye morphological structure and function examination, analysis is comprehensive, and the constructed model is stable, reliable, good in uniformity, long in maintenance time and easy to copy.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models for human diseases, and particularly relates to a method for constructing and evaluating a porcine retinitis pigmentosa disease model. Background Art

[0002] Retinitis pigmentosa (RP) is a group of hereditary retinal diseases mainly involving the retinal pigment epithelium and photoreceptor cells. Clinically, it is mainly manifested as chronic progressive night blindness, visual field constriction, and vision loss. Finding an ideal animal model is of great practical significance for the research and treatment of RP, as well as the development, screening, evaluation, and treatment research of therapeutic drugs.

[0003] Except for blood vessels and the macula, the porcine eye is highly similar to the human eye in terms of morphological size, retinal layer structure, cone cell density, and the ratio of cone cells to rod cells, and has been increasingly widely used in the research of neuroretinal diseases. However, at present, there is a lack of a stable and reliable method for constructing a porcine retinitis pigmentosa disease model. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for constructing and evaluating a porcine retinitis pigmentosa disease model, and the construction method of the present invention can obtain a stable and reliable porcine retinitis pigmentosa disease model.

[0005] The present invention provides a method for constructing and evaluating a porcine retinitis pigmentosa disease model, comprising the following steps:

[0006] Administer iodoacetic acid to the vein of a miniature pig to construct a porcine retinitis pigmentosa disease model;

[0007] After administering iodoacetic acid to the vein of the miniature pig, perform ophthalmological examinations on the miniature pig;

[0008] The ophthalmological examinations include examinations of the eye morphological structure and function.

[0009] Preferably, the miniature pig includes the Bama miniature pig; the miniature pig is a male miniature pig.

[0010] Preferably, the age of the miniature pig is 2.5 - 3.5 months old.

[0011] Preferably, the vein includes the marginal ear vein.

[0012] Preferably, the number of administrations of iodoacetic acid is 1 time; the administration method of iodoacetic acid includes injection.

[0013] Preferably, the concentration of iodoacetic acid is 50 mg / mL.

[0014] Preferably, the dosage of iodoacetic acid is 5-15 mg / kg.

[0015] Preferably, the dosage of iodoacetic acid is 15 mg / kg.

[0016] Preferably, 7 days after iodoacetic acid is administered into the vein of minipigs, photoreceptor cell damage appears in the minipig model of retinitis pigmentosa.

[0017] Preferably, the time for ophthalmic examination includes day 0, 7, 14, 28, 56, and 84 after iodoacetic acid is administered into the vein of minipigs; the ophthalmic examination includes color fundus photography, fluorescein fundus angiography, optical coherence tomography, full-field electroretinogram, and eye histopathology.

[0018] The present invention provides a method for constructing and evaluating a minipig model of retinitis pigmentosa, comprising the following steps: administering iodoacetic acid into the vein of minipigs to construct a minipig model of retinitis pigmentosa; after administering iodoacetic acid into the vein of minipigs, performing ophthalmic examination on the minipigs; the ophthalmic examination includes examination of eye morphological structure and function. The construction method provided by the present invention is a method for constructing a minipig model of retinitis pigmentosa, a non-rodent animal. Using iodoacetic acid as a modeling reagent, it can quickly achieve directional damage to retinal photoreceptor cells within 1 week, with a high modeling success rate. Moreover, after administering iodoacetic acid into the vein of minipigs, the present invention performs ophthalmic examination on the minipigs, including examination of eye morphological structure and function, with comprehensive analysis. The constructed model is stable, reliable, has good homogeneity, long maintenance time, and is easy to replicate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 Color fundus photographs and fluorescein fundus angiograms of each group of animals at 0, 7, 14, 28, 56, and 84 days after modeling;

[0021] Figure 2 Optical coherence tomography images of each group of animals at 0, 7, 14, 28, 56, and 84 days after modeling;

[0022] Figure 3 Thickness change curve graphs of the whole retina layer, inner retina layer (excluding outer nuclear layer), and outer nuclear layer + outer retina layer of each group of animals at 0, 7, 14, 28, 56, and 84 days after modeling;

[0023] Figure 4 For each group of animals, full-field electroretinograms were recorded under dark adaptation at 10.0 for 0, 28, 56, and 84 days after model establishment;

[0024] Figure 5 For each group of animals, full-field electroretinograms were recorded under light adaptation at 3.0 for 0, 28, 56, and 84 days after model establishment;

[0025] Figure 6 For each group of animals, full-field electroretinograms at 30 Hz were recorded under light adaptation for 0, 28, 56, and 84 days after model establishment;

[0026] Figure 7 For each group of animals, graphs showing the changing trends of the amplitudes of the a-wave and b-wave of the full-field electroretinogram under dark adaptation at 10.0 were made for 0, 28, 56, and 84 days after model establishment;

[0027] Figure 8 For each group of animals, graphs showing the changing trends of the amplitudes of the a-wave and b-wave of the full-field electroretinogram under light adaptation at 3.0 were made for 0, 28, 56, and 84 days after model establishment;

[0028] Figure 9 For each group of animals, graphs showing the changing trends of the amplitudes of the full-field electroretinogram at 30 Hz under light adaptation were made for 0, 28, 56, and 84 days after model establishment.

[0029] Figure 10 For each group of animals, the histopathological results of the eye tissues were obtained at 84 days after model establishment. Detailed implementation method

[0030] The present invention provides a method for constructing and evaluating a porcine retinitis pigmentosa disease model, comprising the following steps:

[0031] Iodoacetic acid was administered to the vein of a miniature pig to construct a porcine retinitis pigmentosa disease model; after administering iodoacetic acid to the vein of the miniature pig, ophthalmological examinations were performed on the porcine retinitis pigmentosa disease model; the ophthalmological examinations included examinations of the eye morphological structure and function.

[0032] In the specific implementation process of the present invention, the miniature pig includes the Bama miniature pig, which is a local pig breed in China and has species-specific advantages; the miniature pig is a male miniature pig.

[0033] In the specific implementation process of the present invention, the age of the miniature pig is 2.5 - 3.5 months; the weight of the miniature pig is 9.1 - 12.5 kg, and it is sourced from regular commercial sales.

[0034] In the specific implementation process of the present invention, the vein includes the marginal ear vein.

[0035] In the specific implementation process of the present invention, the number of administrations of iodoacetic acid is 1 time; the administration method of iodoacetic acid includes injection. In the specific implementation process of the present invention, iodoacetic acid is sourced from regular commercial sales.

[0036] In one embodiment of the present invention, the modeling route is a single injection into the auricular vein, which is easy to operate.

[0037] In the specific implementation of the present invention, the concentration of iodoacetic acid is 50 mg / mL.

[0038] In the specific implementation of the present invention, the dosage of iodoacetic acid is 5 to 15 mg / kg.

[0039] In one embodiment of the present invention, the dosage of iodoacetic acid is 15 mg / kg.

[0040] In the present invention, after iodoacetic acid is applied to the vein of a miniature pig for 7 days, photoreceptor cell damage occurs in the pig retinitis pigmentosa disease model, and the modeling rate is high.

[0041] In an embodiment of the present invention, a single injection of 50 mg / mL iodoacetic acid solution at a dose of 15 mg / kg into Bama miniature pigs via the marginal ear vein can cause retinal pigment epithelial atrophy and targeted induction of irreversible apoptosis of photoreceptor cells in the outer nuclear layer and retinal pigment epithelium of the miniature pigs within 1 week, resulting in changes in retinal morphology and function, which can better simulate human retinitis pigmentosa disease, and is a suitable and stable method for rapidly constructing a non-rodent miniature pig retinitis pigmentosa disease model.

[0042] In the specific implementation process of the present invention, the time of the ophthalmological examination includes the 0th, 7th, 14th, 28th, 56th and 84th days after applying iodoacetic acid to the vein of the miniature pig; the ophthalmological examination includes color fundus photography, fluorescein fundus angiography, optical coherence tomography, full-field electroretinogram and ocular tissue pathology.

[0043] In the specific implementation process of the present invention, the concentration of sodium fluorescein injection used in the ophthalmic examination fluorescein fundus angiography is 20%, and the injection dose is 20 mg / kg; the ophthalmic examination optical coherence tomography examination position is about 6 mm just above the edge of the optic disc; the ophthalmic examination full-field electroretinogram examination program is that the miniature pig is fully dilated and dark-adapted for 20 minutes in a dark room environment, and general anesthesia is performed using Shutai combined with xylazine under weak red light conditions, and then the animal is placed on a carrier, and proparacaine is used for surface anesthesia of the ocular surface, and the corneal electrode is dripped with sodium hyaluronate eye drops and then placed on the cornea, the reference electrode is placed under the skin of the outer eyelid margins on both sides, and the ground electrode is placed under the skin of the middle part of the nasal bone between the two eyes, and the impedance between each electrode is kept <10 kΩ, and stimulation is performed with a Ganzfeld full-field stimulator, and dark adaptation 0.01 ERG (flash stimulation intensity and interval are 0.01 cd.sm -2and 2.0 s), dark adaptation 10.0 ERG (flash stimulus intensity and interval are 10.0 cd·s·m -2 and 10.0 s) were detected. After that, light adaptation was performed for 10 min (background light intensity 30.0 cd·m -2 ), and light adaptation 3.0 ERG (flash stimulus intensity and interval are 3.0 cd·s·m -2 and 0.5 s) and light adaptation 30 Hz ERG (flash stimulus intensity and interval are 3.0 cd·s·m -2 and 0.03 s) were detected, and each index was detected 2 times; the eyeballs for ophthalmic examination of eye tissue pathology were fixed with Davidson's fixative, sectioned along the coronal plane, and stained with hematoxylin-eosin (HE staining).

[0044] In the specific implementation process of the present invention, through color fundus photography, no obvious abnormalities were observed in the optic disc, retinal arteriovenous vessels, and retinal pigment epithelium of the modeled animals after modeling.

[0045] In the specific implementation process of the present invention, through fluorescein fundus angiography, no leakage was observed in the main trunks of retinal arteriovenous vessels, but dense mottled fluorescent spots were visible in the peripheral area of the optic disc in the middle and late stages of angiography, indicating atrophy lesions of the retinal pigment epithelium.

[0046] In the specific implementation process of the present invention, the optical coherence tomography included changes in the morphological structure of the retina, and the thickness changes of the entire retinal layer, inner retinal layer (excluding the outer nuclear layer), and outer nuclear layer + outer retinal layer were statistically analyzed.

[0047] In the specific implementation process of the present invention, through optical coherence tomography, it was observed that within 7 days after modeling, the tightness of the connection between the plexiform layer, inner nuclear layer, and outer plexiform layer decreased, the atrophy and thinning of the outer nuclear layer basically disappeared, the retinal pigment epithelium layer was atrophied and thinned in a cloud-like shape, and the IS / OS layer structure was unclear; the thicknesses of the entire retinal layer and the outer nuclear layer + outer retinal layer decreased significantly, and the difference was statistically significant (P < 0.05), while the thickness of the inner retinal layer (excluding the outer nuclear layer) did not change significantly, and the difference was not statistically significant (P > 0.05). Subsequently, as the observation time extended to 84 days, no signs of deepening or recovery of the above lesions were observed.

[0048] In the specific implementation process of the present invention, the full-field electroretinogram includes dark adaptation 10.0, light adaptation 3.0, and light adaptation 30 Hz full-field electroretinograms.

[0049] In the specific implementation process of the present invention, through the full-field electroretinogram, it was observed that the amplitudes of the a-wave and b-wave of dark adaptation 10.0, the a-wave and b-wave of light adaptation 3.0, and the amplitude of light adaptation 30 Hz decreased significantly, and the difference was statistically significant (P < 0.05). No signs of deepening or recovery were observed until 84 days.

[0050] The model evaluation of the present invention uses a combination of multiple indicators, while most of the existing research materials have 1-3 evaluation indicators. Compared with the existing research, the evaluation is more comprehensive and can be used as the evaluation standard for such models. The present invention uses a combination of color fundus photography, fluorescein fundus angiography, optical coherence tomography, full-field electroretinogram, and eye histopathology to conduct a comprehensive evaluation from morphology to visual function and from in vivo to ex vivo. The constructed model is stable, reliable, has good uniformity, a long maintenance time, and is easy to replicate.

[0051] To further illustrate the present invention, the following describes in detail a method for constructing and evaluating a porcine retinitis pigmentosa disease model provided by the present invention in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] Example 1

[0053] 1. Main reagents

[0054] Iodoacetic acid (manufacturer: Shanghai Aladdin Biochemical Technology Co., Ltd.; batch number: H2304197; specification: 98%, AR; expiration date: 2029.03.31)

[0055] 2. Main instruments

[0056] Non-mydriatic fundus camera (model: TRC-NW8F plus, manufacturer: Topcon), laser ophthalmic diagnostic instrument (model: Spectralis OCT, manufacturer: Heidelberg), visual electrophysiology instrument (model: GT-2008V-VI, manufacturer: Guote Medical)

[0057] 3. Experimental animals

[0058] Bama minipigs, 2.5-3.5 months old, 9 animals, single male, weighing 9.1-12.5 kg, purchased from Taizhou Taihe Biotechnology Co., Ltd., quality certificate number: No. 320734241100015583.

[0059] (1) Animal feeding and management

[0060] Animal feeding and management strictly follow relevant SOPs, as well as guidelines or criteria. After the animals are purchased, they are housed in the ordinary environmental animal house with an environmental control system of Guangdong Rhein Medical Research Institute Co., Ltd. The experimental animal use license number is SYXK(Yue)2021 - 0246, issued by the Guangdong Provincial Department of Science and Technology. The environmental temperature in the animal house is 16 - 26°C, the daily temperature difference does not exceed 4°C, the relative humidity is 40% - 70%, the minimum air change rate is ≥8 times / h, the circadian rhythm is 12L:12D (L: light period, D: dark period), the working illuminance is ≥200 Lx, the animal illuminance is 100 - 200 Lx, the noise is ≤60 dB, the air flow velocity at the cage is ≤0.2 m / s, and the ammonia concentration is ≤14 mg / m 3 。The animals are raised in a single - animal mode. The breeding cages are made of stainless steel materials, with the cage size of 1100mm×1100mm×2260mm, and adjacent animals can see and perceive each other.

[0061] (2) Animal welfare

[0062] For the use of animals involved in this example, prior approval has been obtained from the Animal Management and Use Committee (IACUC) of Guangdong Rhein Medical Research Institute Co., Ltd., and the animal experiments are carried out under its supervision.

[0063] 4. Test methods

[0064] (1) Preparation of the modeling reagent: Weigh the iodoacetic acid powder accurately with a thousand -th part balance. Under low - temperature conditions, it is dissolved in 0.9% sodium chloride injection to form a 50 mg / mL solution, assisted by ultrasonic dissolution, and finally filtered and sterilized with a 0.22 -μm water - based microporous membrane under sterile conditions, and stored at - 20°C in the dark for later use.

[0065] (2) Animal modeling: Nine male Bama minipigs are randomly divided into three groups: blank control, 5 mg / kg, and 15 mg / kg, with 3 pigs in each group. Except for the blank control group, the remaining groups are modeled by single - injection of 50 mg / mL iodoacetic acid solution through the marginal ear vein according to the set dose.

[0066] (3) Index detection: Before and after modeling, the animals in each group are sedated and anesthetized, and then color fundus photography, fluorescein fundus angiography, optical coherence tomography, and full - field electroretinogram examinations are carried out; after euthanasia on the 84th day, both eyeballs are taken for histopathological examination.

[0067] (4) Data statistical analysis: SPSS 21.0 software is used for data statistical analysis. One - way ANOVA test is adopted, and a P value < 0.05 is considered to be statistically significant. GraphPad Prism 8 is used for plotting.

[0068] 5. Test results

[0069] (1) Color fundus photography

[0070] Color fundus photographs of each group of Bama minipigs at 0, 7, 14, 28, 56, and 84 days after modeling are as Figure 1 shown. The results show that there were no obvious abnormalities in the optic disc, retinal arteriovenous vessels, and retinal pigment epithelium of the animals in each group before and after modeling.

[0071] (2) Fluorescein fundus angiography

[0072] Fluorescein fundus angiograms of each group of Bama minipigs at 0, 7, 14, 28, 56, and 84 days after modeling are as Figure 1 shown. The results show that there was no leakage in the main trunks of the retinal arteriovenous vessels of the animals in each group. However, in the 15 mg / kg group, dense mottled fluorescent spots were visible in the peripheral area of the optic disc in the middle and late stages of angiography, indicating atrophy of the retinal pigment epithelium.

[0073] (3) Optical coherence tomography

[0074] Optical coherence tomography of each group of Bama minipigs at 0, 7, 14, 28, 56, and 84 days after modeling is as Figure 3 and Figure 4 shown, Figure 3 are the results of optical coherence tomography at 0, 7, 14, 28, 56, and 84 days after modeling, Figure 4 is the thickness change diagram of the whole retina layer, inner retina layer (excluding the outer nuclear layer), and outer nuclear layer + outer retina layer at 0, 7, 14, 28, 56, and 84 days after modeling. The results show that the hierarchical structure of each layer of the retina of the animals in each group was clear and normal at 0 day after modeling. There were no obvious changes in the retinal morphological structure and the thickness of the whole retina layer, inner retina layer (excluding the outer nuclear layer), and outer nuclear layer + outer retina layer of the animals in the blank control and 5 mg / kg groups at 7, 14, 28, 56, and 84 days after modeling compared with those at 0 day after modeling. However, in the 15 mg / kg group, the tightness of the connection between the inner plexiform layer, inner nuclear layer, and outer plexiform layer decreased 7 days after modeling, the outer nuclear layer atrophy became thinner and almost disappeared, the retinal pigment epithelium layer became thinner and showed a cloudy appearance, the IS / OS layer structure was unclear, and at the same time, the thickness of the whole retina layer and outer nuclear layer + outer retina layer decreased significantly, with a statistically significant difference (P < 0.05). There was no obvious change in the thickness of the inner retina layer (excluding the outer nuclear layer), and the difference was not statistically significant (P > 0.05). With the extension of the observation time, by 84 days, there was no sign of deepening or recovery of the above changes.

[0075] (4) Full-field electroretinogram

[0076] Full-field electroretinograms (F-ERG) of each group of Bama minipigs at 0, 28, 56, and 84 days after modeling are as Figures 4 - 9 shown, Figure 4Dark adaptation full-field electroretinogram was performed on animals in each group at 0, 28, 56, and 84 days after modeling for 10.0 Figure 5 Light adaptation full-field electroretinogram was performed on animals in each group at 0, 28, 56, and 84 days after modeling for 3.0 Figure 6 Light adaptation 30Hz full-field electroretinogram was performed on animals in each group at 0, 28, 56, and 84 days after modeling Figure 7 Graph showing the changing trends of the amplitudes of a-wave and b-wave of dark adaptation 10.0 full-field electroretinogram for animals in each group at 0, 28, 56, and 84 days after modeling Figure 8 Graph showing the changing trends of the amplitudes of a-wave and b-wave of light adaptation 3.0 full-field electroretinogram for animals in each group at 0, 28, 56, and 84 days after modeling Figure 9 Graph showing the changing trend of the amplitude of light adaptation 30Hz full-field electroretinogram for animals in each group at 0, 28, 56, and 84 days after modeling. The results showed that there were no abnormalities in the dark adaptation 10.0, light adaptation 3.0, and light adaptation 30Hz of the animals in the blank control group and the 5mg / kg group before and after modeling. However, the a-wave and b-wave of dark adaptation 10.0, the a-wave and b-wave of light adaptation 3.0, and the amplitude of light adaptation 30Hz of the animals in the 15mg / kg group had significantly decreased at 28 days after modeling, and the difference was statistically significant (P < 0.05). No deepening or recovery signs were observed in the above changes until 84 days.

[0077] (5) Ocular histopathology

[0078] The ocular histopathology results of Bama minipigs in each group at 84 days after modeling are as Figure 10 shown. The results showed that the retinal layers of the animals in the blank control group and the 5mg / kg group were clearly distinguishable and the cell connections were tight at 84 days after modeling. However, although the retinal layers of the animals in the 15mg / kg group were also clearly distinguishable, the cells in the ONL layer and PRE layer were arranged in a single layer and were sparse, and the thickness of the photoreceptor cell layer was significantly decreased.

[0079] 6. Conclusion

[0080] Under the conditions of this experiment, after a single intravenous injection of 50mg / mL iodoacetic acid solution at a dose of 15mg / kg into the auricular vein of Bama minipigs, retinal pigment epithelial atrophy and irreversible apoptosis of photoreceptor cells in the outer nuclear layer and retinal pigment epithelial layer of minipigs can be induced within 1 week, resulting in changes in retinal morphology and function, which can better simulate human retinitis pigmentosa disease. It is an appropriate and stable method for rapidly preparing a non-rodent animal minipig retinitis pigmentosa disease model.

[0081] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A method for constructing and evaluating a porcine retinitis pigmentosa disease model, characterized in that: The following steps are involved: Iodoacetic acid was administered into the vein of miniature pigs to establish a pig retinitis pigmentosa disease model. After iodoacetic acid was applied to the miniature pigs’ veins, ophthalmological examinations were performed on the miniature pigs; The ophthalmological examination includes examination of eye morphology, structure and function.

2. The construction and evaluation method according to claim 1, characterized in that: The miniature pig includes Bama miniature pig; the miniature pig is a male miniature pig.

3. The construction and evaluation method according to claim 1, characterized in that: The miniature pig is 2.5 to 3.5 months old.

4. The construction and evaluation method according to claim 1, characterized in that: The veins include the marginal auricular vein.

5. The construction and evaluation method according to claim 1, characterized in that: The iodoacetic acid is administered once; and the iodoacetic acid is administered by injection.

6. The construction and evaluation method according to claim 1, characterized in that: The concentration of the iodoacetic acid is 50 mg / mL.

7. The construction and evaluation method according to claim 1, characterized in that: The dosage of iodoacetic acid is 5-15 mg / kg.

8. The construction and evaluation method according to claim 7, characterized in that: The administration dose of iodoacetic acid was 15 mg / kg.

9. The construction and evaluation method according to claim 1, characterized in that: Photoreceptor cell damage occurred in the porcine retinitis pigmentosa model 7 days after iodoacetic acid was applied to the vein of miniature pigs.

10. The construction and evaluation method according to claim 1, characterized in that: The ophthalmological examinations were performed on days 0, 7, 14, 28, 56 and 84 after intravenous administration of iodoacetic acid to miniature pigs; the ophthalmological examinations included color fundus photography, fluorescein fundus angiography, optical coherence tomography, full-field electroretinogram and ocular histopathology.

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