CN111869625A - High-fat high-sugar diet-induced dry age-related macular degeneration pigment rabbit model - Google Patents
High-fat high-sugar diet-induced dry age-related macular degeneration pigment rabbit model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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Abstract
The invention discloses a construction method of a high-fat high-sugar diet induced dry age-related macular degeneration pigment rabbit model, and belongs to the field of ophthalmic disease animal models. The method of the invention comprises the following steps: adding 10 wt% of lard, 35 wt% of sucrose and 0.5 wt% of cholesterol into the blue-green purple rabbit maintenance feed, and continuously feeding the blue-green purple rabbit for 24 weeks. The method can enable animals to generate dry age-related macular degeneration symptoms only by diet control, is simple and convenient, and is easy for industrial application.
Description
Technical Field
The invention belongs to the field of ophthalmic disease animal models.
Background
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed areas above 50 years. AMD has also become an important blinding eye disease in China as the population ages more and more. The unclear pathogenesis is always the key point and the difficulty of the clinical work of ophthalmology. AMD is classified according to its clinical pathological features as dry (atrophic) and wet (exudative) AMD, the former being characterized by early drusen, changes in the Retinal Pigment Epithelium (RPE) and late geographic atrophy involving the center of the macula; the latter is characterized by Choroidal Neovascularization (CNV). Dry AMD also progresses later to wet AMD, severely impairing the vision of patients, with dry AMD accounting for about 90% of patients clinically. Despite the great advances in the diagnostic and therapeutic studies (e.g., anti-VEGF therapy) for wet AMD, there are no effective treatments or preventatives to date for dry AMD. In addition, due to the medical ethical limitation, most researches on the retina choroid structure, function and biochemistry of AMD cannot directly take human as a research object, the clinical specimen is difficult to obtain, and the dry AMD becomes the bottleneck of AMD research, so that an appropriate animal disease model needs to be selected to guide the research, the pathogenesis of the dry AMD is explored by simulating the structure, the function and the metabolic characteristics of the dry AMD, and a novel treatment is developed so as to prevent or delay the dry AMD progress.
The current dry AMD animal models mainly comprise gene knockout models, drug induction models, photodamaged retina models and the like.
The transgene/gene editing model mainly aims at gene knockout of AMD related genes (such as monocyte chemotactic factor 2 and apolipoprotein E), but has long period, low molding rate, high requirement on personnel equipment and is not suitable for popularization and application.
The drug induction model is prepared by injecting sodium iodate, polyethylene glycol and other drugs into eyes, has high technical requirements on personnel, is easy to cause irreversible acute damage to retina, and is not suitable for AMD model research.
The light damage model is that blue light is used to irradiate animal, and after the light enters the eye, the light can induce the photosensitive molecules of rhodopsin, lipofuscin and the like to generate active oxygen, so that the lipid of the outer segment disc membrane of the photosensitive cell is overoxidized, the apoptosis of the photosensitive cell is induced, and the damage is caused to the retina. The method needs a precise and controllable blue light generating device and has higher requirements on equipment.
The main flow method has the problems of long period, high requirements on personnel and equipment, serious damage to animals and the like, and is not beneficial to industrial application.
The addition of fat and sugar to the animal's diet (i.e., "high-fat high-sugar diet") has been considered a simple and feasible method for modeling dry AMD. The use of a high fat and high sugar diet by the inventors did not induce dry AMD symptoms in all animals. The inventor uses 8-13 years old male aged rhesus as a subject in the early period, and finds that no significant change of choroid and retinal pigment epithelium is seen after the adult rhesus is fed for 18-24 months, namely the adult rhesus cannot be successfully modeled. CN107787912A discloses a dry AMD molding method, which is to mix sucrose, lard, cholesterol and cholate according to the mass ratio of 5-15: 0.5-1.5: 0.1-1.0 to prepare a semi-liquid diet, perfuse the diet once a day through a nasogastric tube (the formula must be perfused manually, otherwise, the monkey does not ingest a certain amount of feed), perfuse 9.2-17.2 g/kg of body weight every day for 15 months. The method has long period and complicated manual perfusion operation, and is not suitable for industrial application.
Disclosure of Invention
The invention aims to solve the problems that: provides a simple and effective construction method of dry AMD animal model.
The technical scheme of the invention is as follows:
the invention provides a method for constructing a rabbit model of dry macular degeneration pigment, which comprises the following steps:
the pigment rabbit takes 8-18 g of lard, 33-55.5 g of cane sugar and 0.3-1.05 g of cholesterol every day for 12-30 weeks.
Further, the construction method comprises the following steps:
10-15 g of lard, 35-52.5 g of cane sugar and 0.5-0.75 g of cholesterol are taken into the pigment rabbit every day for 12-30 weeks.
Further, the lard, the sucrose and the cholesterol are respectively added into a rabbit maintenance feed according to the weight ratio of 10% +/-2%, 35% +/-2% and 0.5% +/-0.2% to prepare a high-fat high-sugar feed, the pigment rabbits are fed with the high-fat high-sugar feed, and 100-150 g of the high-fat high-sugar feed is fed every day;
preferably, the lard, sucrose and cholesterol are respectively added into rabbit maintenance feed according to the weight ratio of 10%, 35% and 0.5%;
further preferably, the feeding time is 24 weeks.
Further, the rabbit maintenance feed contains:
20-27% (w/w) protein, 8-14% (w/w) fat and 60-70% (w/w) carbohydrate;
preferably, the rabbit maintenance feed contains:
23.5% (w/w) protein, 11% (w/w) fat and 65.5% (w/w) carbohydrate.
Further, the pigment rabbit is a blue purple blue rabbit; and/or, the model is dry age-related macular degeneration.
Further, the feeding method comprises the following specific steps: feeding each rabbit 40-60g in the morning and 60-90g in the afternoon.
The invention also provides application of the model obtained by the method in drug screening, wherein the drug is used for treating early age-related macular degeneration.
The invention also provides rabbit feed for constructing an age-related macular degeneration pigment rabbit model, which is prepared by adding 10% +/-2% lard, 35% +/-2% sucrose and 0.5% +/-0.2% cholesterol in weight ratio into rabbit maintenance feed;
the rabbit maintenance feed contains:
20-27% (w/w) protein, 8-14% (w/w) fat and 60-70% (w/w) carbohydrate.
Preferably, the rabbit maintains the added lard content in the feed at 10% (w/w);
and/or, the added sucrose content in the maintenance feed is 35% (w/w);
and/or the content of cholesterol added in the maintenance feed is 0.5% (w/w).
Preferably, the rabbit maintenance feed contains:
23.5% (w/w) protein, 11% (w/w) fat and 65.5% (w/w) carbohydrate.
The term "pigmented rabbit" refers to a rabbit species containing pigmented eyes (i.e., non-albino rabbits).
The invention has the beneficial effects that:
the invention takes the pigment rabbit blue-purple blue rabbit as a target, and the blue-purple blue rabbit can successfully show dry AMD symptom only by feeding high-fat high-sugar diet for 24 weeks, which is unexpected. The method overcomes the problems of long period, high requirement on personnel and equipment, excessive damage to animals and the like of methods such as a gene knockout model, a drug induction model, a photodamage retina model and the like, does not need artificial perfusion to intervene feeding, and is very quick and convenient.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: serum Total Cholesterol (TC), Triglycerides (TG), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL) levels were compared.
FIG. 2: blood glucose level; comparison of blood glucose levels between the left, high-fat high-sugar group (HFSD) and the control group; right, AUC values of IVGTT between high fat high sugar group (HFSD) and control group.
FIG. 3: fundus dazzle color photography (Multicolor), optical coherence tomography (SD-OCT), and autofluorescence detection maps; scale 500 μm.
FIG. 4: fluorescein Angiography (FFA) detection maps; scale 500 μm.
FIG. 5: HE staining, toluidine blue staining and immunofluorescence staining results.
FIG. 6: electroretinogram (ERG) assay results.
Detailed Description
Example 1 model construction
1. Method of producing a composite material
1.1 modeling
Adult male blue-green rabbits (12) with 12-16 weeks old and 3.5 + -0.5 kg body weight were divided into two groups, and the control group (ND) was fed with rabbit maintenance feed (Beijing, Australian cooperative feed Co., Ltd.), and the high-fat high-sugar group (HFSD) was fed with high-fat high-sugar feed in an amount of 40-60g in the morning and 60-90g in the afternoon.
The nutrient components of the maintenance feed are as follows: 23.5% (w/w) protein, 11% (w/w) fat, 65.5% (w/w) carbohydrate; energy: 2.7 kcal/g.
The high-fat high-sugar feed is prepared by adding 10% of lard, 35% of sucrose and 0.5% of cholesterol in weight ratio on the basis of maintaining the feed.
1.2 detection
1) Blood examination
Detection time: before modeling and 8, 12, 16, 24 weeks after modeling;
detection indexes are as follows: blood Glucose (GLU), total Cholesterol (CHOL), Triglycerides (TG), free fatty acids (NFFA), High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL)
2) Glucose Tolerance Test (IVGTT)
Measuring time: 24 weeks after modeling;
the determination method comprises the following steps: fasting was performed for at least 12 hours before sampling and testing without water deprivation. Each rabbit was injected with 1g/kg of glucose (administration volume: 2mL/kg, administration concentration: 500mg/mL) intraperitoneally, 10. mu.L of blood was taken by puncture of the ear margin vein at 0, 0.5, 1.5, and 2 hours after injection, and then the blood was aspirated and tested by a Luo's glucometer (excellent type) siphon chip.
3) Ophthalmic examination
Detection time: before modeling and 24 weeks after modeling;
detecting items: fundus oculi flare photography (multicolor), fluorescein angiography (FFA), fundus autofluorescence, optical coherence tomography (SD-OCT).
4) Histopathological examination of the eyeball
Detection time: week 24.
Each group of rabbit eyes was removed, punctured with a small incision along the limbus, and placed in modified Davidson's fixative 4 degrees overnight for paraffin section preparation.
Pathological staining: sections from each group were subjected to hematoxylin-eosin (HE), toluidine blue and immunofluorescence staining (for Bm3 and IB4 proteins), parallel microscopy and counting analysis.
5) Electroretinogram (ERG) detection
Detection time: week 24.
Animals were individually dark-adapted for 30min and stimulated with international clinical visual electrophysiological standard parameters, and dark-adapted ERGs (including Scotopic 0.01ERG, Scotopic 3.0 OPS) were recorded using routine procedures, and then light-adapted ERGs (Photopic 3.0 flickers) were recorded after 10min of animal light adaptation.
2. Results
2.1 blood test results
In general, the total cholesterol, triglyceride and low-density lipoprotein in the high-fat and high-sugar group serum are obviously increased 60 days after the high-fat and high-sugar feed is fed, and the high-density lipoprotein is also increased to a certain extent but is slightly increased (figure 1).
Blood glucose values in the high-fat and high-sugar group were significantly higher than those in the control group from day 120, and AUC values in IVGTT at day 180 were also significantly higher than those in the control group (fig. 2).
The results show that the feeding method can improve the blood fat and blood sugar of the blue and green rabbits.
2.2 results of ophthalmologic examination
Fundus oculi glare photographs showed a decrease in the nerve fiber bundles around the optic papilla in the high-fat and high-sugar group (fig. 3, Multicolor); optical coherence tomography showed a discontinuity in the RPE (retinal pigment epithelium) layer with a finger-like bulge in the lower part of the RPE layer (fig. 3, indicated by the arrow in the OCT column); autofluorescence showed strong fluorescence under RPE as lipid-like material deposition (fig. 3, indicated by autofluorescence bar arrows), visible generation of Drusen (Drusen) -like deposits.
Fluorescein angiography results showed vascular leakage around the optic papilla in the high-lipid high-sugar group and neovascular-like leakage at the ends of the vessels (fig. 4).
Among the above results, decreased nerve fiber bundles may be associated with age-induced AMD and retinal nerve fiber loss; RPE digital protrusions and lipid deposition under RPE are consistent with RPE-altering symptoms of dry AMD, since rabbits lack of macular area, peripapillary vascular leakage may be associated with breakdown of the extraretinal barrier and high-fat high-sugar induced retinal ischemia hypoxia leading to vascular proliferation, similar to the transition of dry AMD to wet AMD leading to neovascularization into the retina.
2.3 examination of the histopathology of the eyeball
Both HE and toluidine blue staining showed lipid-like deposition under the RPE (fig. 5, yellow/red arrows in the HE and toluidine blue bars) and malformation and degeneration of photoreceptor outer segments (fig. 5, toluidine blue bar, white arrows). Immunofluorescence results show that the central retina Brn3 positive ganglion cells in the high-fat high-sugar group are obviously less than those in the control group, and the total area of IB4 positive blood vessels is larger than that in the control group.
The pathological results of the part show that the deposition of lipoid substances under the RPE is identical with the deposition of lipofuscin under the RPE of dry AMD; meanwhile, photoreceptor degeneration is consistent with damage of the outer nuclear layer of dry AMD, and the degeneration can further influence the activity of ganglion cells due to aging; furthermore, vascular proliferation of the retina (shown by the staining results of IB 4) may be similar to that of dry AMD.
2.4ERG
ERG results show that the amplitude of dark-adapted b-wave in the high-fat and high-sugar group is obviously reduced compared with the control group, and the b-wave latency period is also prolonged compared with the control group.
The results show that: the amplitude of the dark-adapted b wave and the long and short latency represent the functional state of the outer nuclear layer of the retina, the amplitude of the dark-adapted b wave of the high-fat and high-sugar group is obviously reduced compared with that of the control group, and the prolonged latency of the b wave indicates that the function of the outer nuclear layer of the retina is damaged, which is consistent with the function reduction of the outer nuclear layer (including photoreceptors) of the dry AMD.
In conclusion, the blue-green rabbits are taken as the target, and the blue-green rabbits can successfully show dry AMD symptoms only by feeding high-fat high-sugar diet for 24 weeks. The method overcomes the problems of long period, high requirement on personnel and equipment, excessive damage to animals and the like of methods such as a gene knockout model, a drug induction model, a photodamage retina model and the like, does not need artificial perfusion to intervene feeding, and is very quick and convenient.
Claims (10)
1. A construction method of a rabbit model of dry macular degeneration pigment is characterized by comprising the following steps:
the pigment rabbit takes 8-18 g of lard, 33-55.5 g of cane sugar and 0.3-1.05 g of cholesterol every day for 12-30 weeks.
2. The method of construction of claim 1, wherein: it includes:
10-15 g of lard, 35-52.5 g of cane sugar and 0.5-0.75 g of cholesterol are taken into the pigment rabbit every day for 12-30 weeks.
3. The method of construction of claim 1, wherein:
adding the lard, the sucrose and the cholesterol into a rabbit maintenance feed according to the weight ratio of 10% +/-2%, 35% +/-2% and 0.5% +/-0.2% respectively to prepare a high-fat high-sugar feed, feeding the pigment rabbits, and feeding 100-150 g of the high-fat high-sugar feed every day;
preferably, the lard, sucrose and cholesterol are respectively added into rabbit maintenance feed according to the weight ratio of 10%, 35% and 0.5%;
further preferably, the feeding time is 24 weeks.
4. The method of construction of claim 3, wherein:
the rabbit maintenance feed contains:
20-27% (w/w) protein, 8-14% (w/w) fat and 60-70% (w/w) carbohydrate;
preferably, the rabbit maintenance feed contains:
23.5% (w/w) protein, 11% (w/w) fat and 65.5% (w/w) carbohydrate.
5. The method of claim 1, wherein: the pigment rabbit is a blue purple rabbit; and/or, the model is dry age-related macular degeneration.
6. The method of any of claims 1 to 5, wherein: the feeding method comprises the following specific steps: feeding each rabbit 40-60g in the morning and 60-90g in the afternoon.
7. Use of the model obtained by the method of any one of claims 1 to 6 in screening for a drug for the treatment of early age-related macular degeneration.
8. A rabbit feed for constructing an age-related macular degeneration pigment rabbit model, which is characterized in that:
the rabbit feed is prepared by adding 10% +/-2% lard, 35% +/-2% sucrose and 0.5% +/-0.2% cholesterol in the rabbit maintenance feed by weight ratio;
the rabbit maintenance feed contains:
20-27% (w/w) protein, 8-14% (w/w) fat and 60-70% (w/w) carbohydrate.
9. The rabbit feed of claim 8, wherein:
the rabbit maintains the lard content added in the feed to be 10% (w/w);
and/or, the added sucrose content in the maintenance feed is 35% (w/w);
and/or the content of cholesterol added in the maintenance feed is 0.5% (w/w).
10. The rabbit feed according to claim 8 or 9, wherein: the rabbit maintenance feed contains:
23.5% (w/w) protein, 11% (w/w) fat and 65.5% (w/w) carbohydrate.
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