CN103720524A - Method for making primate dry age-related macular degeneration disease model - Google Patents
Method for making primate dry age-related macular degeneration disease model Download PDFInfo
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- CN103720524A CN103720524A CN201410012503.0A CN201410012503A CN103720524A CN 103720524 A CN103720524 A CN 103720524A CN 201410012503 A CN201410012503 A CN 201410012503A CN 103720524 A CN103720524 A CN 103720524A
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Abstract
The invention discloses a method for making a dry age-related macular degeneration disease model on a primate, namely applying sodium iodate onto the primate with a carotid artery injection method. The method is simple and easy, high in success rate and good in repeatability. The model made with the method is stable. The amount of sodium iodate needed is small, injury of large doses of sodium iodate to the whole body of the animal is avoided, and the animal can live for a long time so that follow-up tests can be made possible. A model of any one side or a model of double sides can be made as needed. When the one-side model is made, a lateral eye can not be hurt at all, and normal living vision of the animal is guaranteed. Comparison of two lateral eyes of one animal can be conducted to provide credibility for the test, and the number of animals needed for the test is reduced. The method provides a sound tool for the dry age-related macular degeneration research and has great significance for the pathological physiology research and treatment research of the disease.
Description
Technical field
The present invention relates to field of medical technology, specifically a kind of method of making primate Local Electroretinogram disease model.
Background technology
Local Electroretinogram (age-related macular degeneration, AMD) is to betide one of common blinding oculopathy in middle-aged and elderly people.Research shows retinal pigment epithelium (retinal pigment epithelium, RPE) normal function has vital meaning to the quantity of photoreceptor cell，photosensory cell and function, in AMD generating process, the infringement of macular area retinal pigment epithelium is the primary affection of disease, and the loss of photoreceptor cell，photosensory cell is secondary.With age, AMD sickness rate rises gradually, so its harm just increases along with the aging of China's population.Dryness senile degeneration of macula is now still without clear and definite effectively treatment means, and its pathogeny and Therapeutic Method become study hotspot, but because there is no desirable animal disease model, have hindered greatly its progress.
Sodium iodate is a kind of antimetabolite, the activity of can destroying retinal pigment epithelium carbohydrate metabolism relevant enzyme, thereby can selective destruction retinal pigment epithelium.By intravenous injection sodium iodate, in rabbit, can produce Local Electroretinogram model.Primate is the most similar to mankind's 26S Proteasome Structure and Function, the disease model of primates is very important to the research of human diseases, but in primate, the sodium iodate intravenous injection of corresponding dosage can not obtain satisfied Local Electroretinogram model, increase dosage and increase the general toxicity of medicine, animal is even because medicine general toxicity is dead.The progress > > (Jiang Wei of document < < age-related macular degeneration (AMD) experimental model, Qiu Chunyi, international ophthalmology magazine > > the 3rd phase in 2007 of < <) and document < < sodium iodate induce the research > > (Jiang Wei of non-exudative type age-related macular degeneration model, Zhang Wanyu, Qiu Chunyi, international ophthalmology magazine > > the 8th phase in 2008 of < <) disclosing the mode that adopts sublingual vein to inject is that 30-40mg/kg sodium iodate is for modeling in the body of non-exudative type age-related macular degeneration by dosage.Method due to the modeling of intravenous injection sodium iodate, medicine first arrives heart through venous return, after through heart, pump and pass through aorta again, carotid artery, ophthalmic artery enters retina choroidal circulation, medicine significantly reduces through systemic circulation the drug level that arrives eye, and medicine is concentration dependent to the effect of pigment epithelium, in order to reach the object of destroying pigment epithelium, strengthen dosage, but general toxicity that like this can corresponding increase medicine causes animal hematuria, renal failure is even dead, and the pathological changes of corresponding retinal pigment epithelium not obvious.
Summary of the invention
The present invention is directed to prior art deficiency, a kind of disease model of primate Local Electroretinogram of satisfaction is provided.By through carotid artery administration, medicine to ophthalmic artery, enters retina choroidal circulation through carotid artery, and medicine does not pass through the dilution of systemic circulation, and the drug level that arrives eye is high, thereby uses a small amount of medicine and reach corresponding object; Can also control the time that medicine stops at eye further by controlling carotid Xining, the effect of prolong drug to pigment epithelium, can regulate and control scope and degree that retinal pigment epithelium damages.
The object of the invention is to solve by the following technical programs:
Make a method for primate Local Electroretinogram disease model, sodium iodate is imposed on to primate in the mode of carotid injection, the dosage of above-mentioned sodium iodate is 15mg/kg.
Applicant finds, in the manufacturing process of primate Local Electroretinogram disease model of the present invention, the degree of modelling effect and pathological changes not only has direct relation with the dosage of sodium iodate, also relevant with sodium iodate drug level and the speed of injecting, preferably the concentration of sodium iodate is 10mg/ml, and the speed of injecting is 0.1ml/s.
In said method, carotid injection can adopt the mode administration of carotid puncture.When injecting, the general horizontal position that adopts, animal cervical region is padded, head is to layback, expose cervical region as far as possible, head is partial to the offside of site of puncture, carry out after conventional skin degerming, with left index finger or forefinger, middle finger, fix its obviously blood vessel at place of beating, the fixing accurate needle handle of right hand forefinger and thumb position, entry point is below thyroid cartilage lower edge, during inserting needle, will note inserting needle direction, its vertical angle is to be as the criterion with carotid artery vertical direction, after thrusting, there is pulsation blood to spray in needle tubing, get final product injection of medicine.The complete hemostasis by compression of drug injection, can block common carotid artery certain hour by further pressing, thereby further extend the time that sodium iodate is assembled at eye, and preferably Xining is 1 minute.
Or, can adopt the carotid mode of exposure to inject: 1) by Animal Anesthesia, fixing, expose cervical region, unhairing, sterilization; 2) throat's median line skin incision, exposes common carotid artery; 3) blocking-up external carotid artery and common carotid artery blood flow; 4) sodium iodate is injected through common carotid artery, continue to keep common carotid artery bloodstream blocking certain hour, preferably 1 minute.5) pull out hemostasis by compression after pin, sew up the incision.
The present invention has the following advantages compared to existing technology:
1) modeling success rate is high, favorable repeatability, model stability;
2) required sodium iodate medication amount is few, has avoided the whole body damage of heavy dose of sodium iodate to animal, and Long term Animal survival, for follow-up test provides possibility;
3) can do as required any side or bilateral model;
4) while doing a side modeling, Second eye is not had to any damage, animal has vision orthobiosis, has guaranteed animal welfare;
5) can on an animal, do the own control of bilateral eye, the credibility of test is provided, reduce the requirement of experimental animal.
The present invention, for Local Electroretinogram provides a good instrument, studies significant to the pathophysiology research of this disease and treatment.
Accompanying drawing explanation
Fig. 1 is six weeks eyes dark adaptation 3.0 electroretinograies after the modeling of machin 15mg/kg group sodium iodate (the maximum hybrid reaction of rod cell cone cell, has reacted the function of retinal rod and cone cell, and left eye is modeling eye).
Fig. 2 is six weeks eyes light adaptation 3.0 electroretinograies after the modeling of machin 15mg/kg group sodium iodate (cone responses, has reacted the function of macular area cone cell, and left eye is modeling eye).
Fig. 3 is eyes dark adaptation 3.0 electroretinograies before and after the modeling of machin various dose sodium iodate (the maximum hybrid reaction of rod cell cone cell) a wave-amplitude change curves.
Fig. 4 is eyes light adaptation 3.0 electroretinograies (rod cell reaction) a wave-amplitude change curve before and after the modeling of machin various dose sodium iodate.
Fig. 5 is six weeks eyes fundus photographs after the modeling of machin 15mg/kg sodium iodate, and left eye is modeling eye.
Fig. 6 is six weeks eyes autofluorescence photos after the modeling of machin 15mg/kg sodium iodate, and left eye is modeling eye.
Fig. 7 is six weeks eyes OCT results after the modeling of machin 15mg/kg sodium iodate, and left eye is modeling eye.
The specific embodiment
Concrete steps of the present invention are described by the following examples, but not limited by embodiment.
The term that used in the present invention, except as otherwise noted, generally has the implication that those of ordinary skills understand conventionally.
Below in conjunction with specific embodiment comparable data, the present invention is described in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following examples, various processes and the method do not described in detail are conventional methods as known in the art.
Embodiment 1 carotid injection sodium iodate is set up machin Local Electroretinogram model.
Object: set up machin Local Electroretinogram model by carotid injection sodium iodate, filter out the appropriate dose of modeling.
Experiment material: main agents:
Narcotics: 50mg/ml ketalar, 3% pentobarbital sodium
Disinfection drug: 5.0g/L povidone iodine
Modeling medicine: sodium iodate (Sigma company, lot number: S4007), under the environment of darkroom, with 0.9% sterile saline, sodium iodate powder is melted to the concentration to 10mg/ml, and filter with 0.22 μ m filter disc.
Fundus fluorescein angiography agent: 500 milligrams/5 mls/branch of fluorescein sodium injection (vertical take the photograph)
Key instrument:
YZ5F slit lamp microscope (Hangzhou six or six)
Espion visual electrophysiology instrument (U.S. Diagnosys company)
SPECTRALIS OCT instrument (Heidelberg, Germany company)
Heidelberg fluoroscopic visualization machine, fundus camera
Laboratory animal:
Animal feeding is carried out according to SOP with management, follows experimental animal feeding and guide for use the 8th edition (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council simultaneously; National Academy Press, Washington, D.C., 2010) and United States Department of Agriculture (USDA) (USDA) animal welfare management rules (Public Law99-198).The use of laboratory animal has obtained the approval of Jiangsu Province Science and Technology Department, laboratory animal occupancy permit number: SYXK(Soviet Union) 2011-0029.
Machin is zoopery conventional animal, and physiological characteristics is similar to people, and it is convenient to raise, and is convenient to test operation.
From deposit, select 6 of the normal machins of general health check-up, blood biochemistry and ophthalmic system inspection animal, 2-5 year when administration starts, body weight 2-5kg.On Animal neck ring, No. ID, marking animals, and simultaneously labelling experiment animal number is as the sign of animal.The corresponding relation of Record ID number and animal number.
After sodium iodate injection modeling, in one week, take single cage to raise, all the other times are taked group support cage feeding manner.Animal House design temperature is 18～26 ℃, and humidity is 40～70%, and 12 hours light and shades of illumination alternately.
1. experimental technique:
6 machins, be divided into tri-groups of 10mg/kg, 15mg/kg, 20mg/kg, every group each two, respectively each group machin left common carotid injection is carried out to the sodium iodate administration of corresponding dosage, with left eye modeling, autologous right eye is not modeling contrast, after administration, weekly each dosage group is carried out to eyes retina electrograph (Electroretinogram, ERG), fundus photography, fundus autofluorescence, OCT inspection.
In said method, intra-arterial injection adopts the mode administration of carotid puncture: when injecting, adopt horizontal position, animal cervical region is padded, head is to layback, expose cervical region as far as possible, head is partial to the offside of site of puncture, carry out after conventional skin degerming, with left index finger or forefinger, middle finger is fixed its obviously blood vessel at place of beating, the fixing accurate needle handle of right hand forefinger and thumb position, entry point is below thyroid cartilage lower edge, during inserting needle, to note inserting needle direction, its vertical angle is to be as the criterion with carotid artery vertical direction, after thrusting, there is pulsation blood to spray in needle tubing, get final product injection of medicine (injection speed is 0.1ml/s).The complete hemostasis by compression of drug injection, presses blocking-up common carotid artery 1 minute, thereby further extends the time that sodium iodate is assembled at eye.
Or also can select the carotid mode of exposure to inject: 1) by ketamine for animal (0.2ml/kg) intramuscular injection, and pentobarbital sodium (20mg/kg) hind leg intravenous injection anesthesia, face upward and be fixed on operating-table, expose cervical region, unhairing, sterilize with iodophor solution in part; 2) throat's median line skin incision, exposes common carotid artery; 3) blocking-up external carotid artery and common carotid artery blood flow; 4) sodium iodate is injected to (injection speed is 0.1ml/s) through common carotid artery crotch, continue to keep common carotid artery bloodstream blocking 1 minute; 5) pull out hemostasis by compression after pin, sew up the incision.
Above-mentioned two kinds of methods all can make machin Local Electroretinogram model, no significant difference.
Experimental result:
(1) eyes retina electrograph (Electroretinogram, ERG) check result
ERG check result is shown in Fig. 1-4, Fig. 1 is six weeks eyes dark adaptation 3.0ERG after the modeling of machin 15mg/kg group sodium iodate, left eye is modeling eye, right eye is that (figure a and figure b are dark adaptation 3.0ERG to autologous not modeling contrast eye, figure c and figure d are dark adaptation 3.0 oscillation potential ERG, wherein scheming a and scheming c is right eye, and figure b and figure d are left eye), result shows that left eye a wave-amplitude slightly declines compared with right side; Fig. 2 is six weeks eyes light adaptation 3.0ERG after the modeling of machin 15mg/kg group sodium iodate, and left eye is modeling eye, and right eye is autologous not modeling contrast eye (left figure is right eye, and right figure is left eye), and result shows that left eye a wave-amplitude is about 1/3rd of right side; Fig. 3 is eyes dark adaptation 3.0ERG a wave-amplitude change curve before and after the modeling of machin various dose sodium iodate; Fig. 4 is eyes light adaptation 3.0ERG a wave-amplitude change curve before and after the modeling of machin various dose sodium iodate.Result shows each dosage group, dark adaptation 3.0, and oscillation potential, its a wave-amplitude of light adaptation 3.0ERG all declines to some extent, all slowly go up, but degree is obviously different after modeling in 6 weeks.
Dark adaptation 3.0ERG is the maximum hybrid reaction of retinal rod cone cell, has reacted the function of retinal rod and cone cell; In the experiment of this group, 10mg/kg group dark adaptation 3.0ERG a wave-amplitude returns to the front level of modeling substantially in the time of 6 weeks, 15mg/kg group returns to modeling the last two level of first three minute, 20mg/kg group return to modeling the first five/level, illustrate that 10mg/kg group is less to the damage of retinal rod cone cell, the damage to some extent of 15mg/kg group, and 20mg/kg is to the larger (see figure 3) of its damage.Light adaptation 3.0ERG is cone responses, reacted the function of cone cell, in the experiment of this group, 10mg/kg group a wave-amplitude returns to the front level of modeling substantially, 15mg/kg and 20mg/kg group return to preoperative 1/3rd horizontal (see figure 4)s, illustrate that 15mg/kg and 20mg/kg treated animal cone cell function have obvious decline.In conjunction with the result of dark adaptation 3.0ERG and light adaptation 3.0ERG, we can find out 15mg/kg group main damage macular area cone cell function, and the damage of 20mg/kg group is not only the function of cone cell, and the function of rod cell is also subject to obvious damage.
(2) fundus photography check result
After the modeling of machin 15mg/kg sodium iodate, as shown in Figure 5, left eye is 15mg/kg sodium iodate modeling group to six weeks eyes fundus photography check results, and autologous right eye is modeling matched group not.Result shows the atrophy of left eye macular area retinal pigment epithelium, mottling atrophy, and the side's of seeing below choroidal artery thoroughly, periphery has no obvious change.The pigment epithelium atrophy of the whole rear utmost point of 20mg/kg group portion.
(3) autofluorescence
After the modeling of machin 15mg/kg sodium iodate, as shown in Figure 6, left eye is 15mg/kg sodium iodate modeling group to six weeks eyes autofluorescence results, and autologous right eye is modeling matched group not.Compared with the control, 15mg/kg group left eye macular area loses the autofluorescence of uniformity and shows the local low fluorescence of the high fluorescence companion of mottling in result demonstration, and the outer fluorescence of macula lutea is even, and damage and the function reduction of macular area pigment epithelium is described.
(4) OCT check result
After the modeling of machin 15mg/kg sodium iodate, as shown in Figure 7, left eye is 15mg/kg sodium iodate modeling group to six weeks eyes OCT check results, and autologous right eye is modeling matched group not.Result shows 15mg/kg group, and before modeling, each layer of structure of retina choroid is normal, macular area pigment epithelium layer atrophy after modeling, and ectoretina structure disturbance, IS/OS layer is invisible.
Discuss: ERG result shows that the sodium iodate carotid injection of 10-20mg/kg can cause the damage of retinal pigment epithelium, wherein 20mg/kg group dark adaptation 3.0 and light adaptation 3.0 amplitudes obviously decline, after six weeks, maintain reduced levels, show that retinal rod and cone cell function all decline, damage be whole rear utmost point portion retina, do not meet the feature of dryness senile degeneration of macula main damage macular area cone cell; Although and 15mg/kg group dark adaptation 3.0ERG amplitude rising after modeling, but it is not obvious that light adaptation 3.0ERG rises, illustrate that rod cell function damage is compared with light and cone cell function damage is serious, the retina injury causing is confined to macular area, meets the feature of the change of Age-related related macular degeneration.The damage limitation that fundus photograph shows 15mg/kg group macular area retinal pigment epithelium and rear utmost point portion macular area retina, approach the Clinical changes of Local Electroretinogram; Autofluorescence and OCT have shown macular area pigment epithelium and ectoretina atrophy on live body, and age-dependent degeneration of macula changes similar.Visible sodium iodate carotid injection can cause the pathological changes of retinal pigment epithelium, the macular area retinal pigment epithelium damage that wherein injection of the sodium iodate of 15mg/kg causes is moderate, similar to the pathological change of people's age-related macular degeneration, to the not obviously damage of animal whole body, be a suitable modeling dosage.
Claims (3)
1. make a method for primate Local Electroretinogram disease model, it is characterized in that sodium iodate to impose on primate in the mode of carotid injection.
2. the method for claim 1, the dosage that it is characterized in that sodium iodate is 15mg/kg.
3. the method for claim 1, the concentration that it is characterized in that sodium iodate is 10mg/ml, the speed of injecting is 0.1ml/s.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107787912A (en) * | 2017-10-20 | 2018-03-13 | 广东省生物资源应用研究所 | A kind of abductive approach of Local Electroretinogram primate model |
| JP2018157809A (en) * | 2017-03-23 | 2018-10-11 | 株式会社浜松ファーマリサーチ | Aging macular degeneration model animal of nonhuman primate and its creation method |
| CN111869625A (en) * | 2020-09-18 | 2020-11-03 | 四川大学华西医院 | High-fat high-sugar diet-induced dry age-related macular degeneration pigment rabbit model |
| CN111990339A (en) * | 2020-09-18 | 2020-11-27 | 四川大学华西医院 | High-sugar diet-induced early dry age-related macular degeneration pigment rabbit model |
-
2014
- 2014-01-10 CN CN201410012503.0A patent/CN103720524A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018157809A (en) * | 2017-03-23 | 2018-10-11 | 株式会社浜松ファーマリサーチ | Aging macular degeneration model animal of nonhuman primate and its creation method |
| CN107787912A (en) * | 2017-10-20 | 2018-03-13 | 广东省生物资源应用研究所 | A kind of abductive approach of Local Electroretinogram primate model |
| CN107787912B (en) * | 2017-10-20 | 2023-04-11 | 广东省科学院动物研究所 | Induction method of primate model with dry age-related macular degeneration |
| CN111869625A (en) * | 2020-09-18 | 2020-11-03 | 四川大学华西医院 | High-fat high-sugar diet-induced dry age-related macular degeneration pigment rabbit model |
| CN111990339A (en) * | 2020-09-18 | 2020-11-27 | 四川大学华西医院 | High-sugar diet-induced early dry age-related macular degeneration pigment rabbit model |
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