CN107981837B - Comparative research method for yangtao axis development of macaca by using illumination light source - Google Patents
Comparative research method for yangtao axis development of macaca by using illumination light source Download PDFInfo
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Abstract
The invention relates to a method for contrast research on the development of an eye axis of a macaque animal by an illuminating light source, which comprises the following steps: selecting a plurality of macaca animals with visual index screening detection values meeting requirements as experimental animals, and randomly distributing the experimental animals into N rearing cages placed in different lighting sources; after feeding for one week, detecting the initial detection value of the eye visual index of each experimental animal, excluding the experimental animals with obvious difference between the initial detection value and the screening detection value, averaging the initial detection values and the screening detection values of the rest experimental animals as basic detection values, and continuously feeding; after raising for 6-24 months, detecting the final detection value of the visual index of each experimental animal; and carrying out comparison analysis according to the basic detection value and the final detection value to obtain analysis data of the influence of different illumination light sources on the vision development of the experimental animal. The invention has no harmful treatment to animals in the experimental process, thereby being a research method which is closer to the development state of natural myopia.
Description
Technical Field
The invention relates to a method for detecting influence of different illuminations on natural development of an eye axis of a young macaque animal in a non-artificial interference state, and belongs to the field of photobiosafety and photobiological effect.
Background
1. Excessive growth of the axis of the eye is the most important physiological manifestation of the occurrence of myopia. When the length of the axis of the eye is larger than the normal value, in the state of adjustment and relaxation, the parallel light rays cannot be focused on the retina after being refracted by the dioptric system of the eyeball, but are converged in front of the retina, so that the distant scene is not clear, and the myopia is caused. This type of myopia, also known as axial myopia, is the most prominent type of myopia among primary and middle school students. It is generally accepted that ocular axis development is influenced by a number of factors, the most closely related to light.
2. The axis of the eye develops most rapidly in vertebrate immature individuals and is most susceptible to external factors. After adult life, the eye axis of the organism is not substantially changed. Recent studies have shown that the development of the axis of the eye and the occurrence of subsequent myopia are greatly affected by the different illumination received by people between 3 and 8 years of age.
3. In the animal experiments of myopia research, the related experimental animals mainly comprise chickens, guinea pigs, big mice, rabbits, macaques and the like. Due to species differences, experimental results obtained from different species of experimental animals are often inconsistent and difficult to popularize in humans. Among them, the rhesus monkey and the human have the most similar visual system and eyeball structure and human height because of the closest relationship. Therefore, the experimental results obtained in macaques can be better extended to humans.
4. In the traditional animal experiments for myopia studies, the age of the experimental animals was mainly new born animals (chicken: experiment started about 7 days after shell breaking; big and small mice: experiment started after birth until eyes are opened; macaque: experiment started about 30 days after birth). The method for inducing myopia mainly comprises the following steps: 1) the eyelid of one eye of the experimental animal is sutured; 2) placing a concave lens (optical defocus model) on one eye of the experimental animal; 3) a diffusion lens (form deprivation model) was placed on one eye of the experimental animal. The traditional methods have the advantage that the excessive growth of the axis of the eye of the experimental animal can be quickly induced (several days to thirty days), so that the experimental progress can be greatly accelerated. However, these conventional methods also have significant disadvantages, mainly: 1) the age bracket of the animal is greatly lower than that of the human suffering from myopia. These animals are still in a rapid developmental stage of the post-neonatal visual system, at which time the onset of myopia experiments may be influenced by a variety of developmental factors; 2) the process of inducing myopia in animals is not consistent with the process of inducing myopia in humans by placing a concave or diffusing lens on the outside of the animal's head or eyes.
5. Patent No. 200620062570.4 discloses an eye patch for inducing a myopia model in a non-human primate such as a monkey. The principle of the eye mask is based on optical defocus and form-deprivation. It is designed in a circular helmet shape, and all the components are fixed by screws, and the lens is positioned in front of the eyeshade. The design is suitable for inducing the myopia of the monkey rapidly through the lens, but the myopia is different from the myopia development process of the monkey or the human in the natural state. And the patent does not measure and correct for the light rays after the lens is deflected and blurred, which would interfere with the interpretation of the possible mechanisms of myopia production.
6. Patent No. 201310238070.6 discloses a glasses wearing system for rabbit, cat and dog to induce myopia and its application method. The mirror-wearing system includes: the truncated cone-shaped mask, the lens support, the induction lens, the fixing belt and the buckle are realized, and the lens can be quickly replaced. The principle of inducing myopia is also based on optical defocus and form deprivation. The design is suitable for rapidly inducing myopia of rabbits, cats and dogs through the lens, but the myopia is different from the myopia development process of people in a natural state. And the patent does not measure and correct for the light after the lens is deflected and blurred, which would interfere with the interpretation of the possible mechanisms of myopia.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for contrast research on the development of the eye axis of an animal of the genus macaque by an illuminating light source, the method does not need to place a lens outside the eyes of an experimental animal, no harmful treatment is carried out on the animal in the experimental process, and the experimental result is closer to the development state of natural myopia.
In order to solve the technical problem, the method for the comparative study of the illumination light source on the development of the axis of eyes of the macaca animal comprises the following steps:
selecting a plurality of healthy macaca animals with ages of 3-24 months, detecting the visual development state of the healthy macaca animals, screening the macaca animals with visual indexes meeting the experimental requirements as experimental animals, and simultaneously recording the visual index screening detection values S of the experimental animalsiI is the number of the experimental animal;
step two, randomly dividing the screened experimental animals into N groups, and respectively placing the N groups of experimental animals in N rearing cages, wherein N is more than or equal to 2, and the number of the experimental animals in each group is not less than 6; then respectively placing N-1 rearing cages in rearing rooms with different illumination light sources to breed the experimental animals; one breeding room is illuminated by natural light;
step three, after raising for one week, detecting the visual development state of the eyes of each experimental animal to obtain the initial visual index detection value S of each experimental animal at the momenti'; excluding the initial detection value Si' and screening test value SiThe experimental animals with obvious difference are subjected to initial detection values S of the other experimental animalsi' and screening test value SiTaking the average as number iBasic detection value Z of visual index of experimental animaliAnd continuing to raise;
step four, after raising for 6-24 months, detecting the visual development state of the eyes of each experimental animal again to obtain the final detection value Z of the visual index of each experimental animali‘；
Step five, according to the basic detection value Z of the visual index of each experimental animaliAnd the final detection value Zi' comparative analysis is carried out, thereby obtaining the analysis data of the influence of different illumination light sources on the visual development of the experimental animal.
The lighting light source comprises two or more than two of an LED lamp, a fluorescent lamp, an incandescent lamp, a halogen lamp and an electrodeless sulfur lamp.
The illumination of the central position in the rearing cage is 200 and 750 lx.
The temperature in the breeding room is controlled between 20 and 25 ℃, the relative humidity is controlled between 40 and 70 percent, and the ventilation frequency is not lower than 8 times per hour.
The visual indicator is primarily eye axis length.
The visual indicators may also include ocular diopters, anterior chamber depth, corneal curvature.
In the third and fourth steps, the spectrum of the illumination light source and the illumination in the rearing cage can be regularly detected, so that the stability of the spectrum and the illumination in the experimental process is ensured; meanwhile, the lighting source replaced in the experiment is reserved as a light source sample for storage at regular intervals.
Furthermore, when the visual indexes of the macaque are detected, the macaque is subjected to conventional anesthesia, and after the macaque does not have pain sensation reaction, the head of the macaque is fixed on the positioning instrument and starts to be detected facing the detection instrument.
If the two eyes of the macaque are closed in the detection process, the upper eyelid and the lower eyelid of the macaque are opened by the eyelid opener, then the examination of each index is carried out, and artificial tears are regularly dropped on the surfaces of the eyes of the macaque in the examination process to ensure the wetting of the surfaces of the eyeballs of the macaque.
The locator comprises a head fixing mechanism, a three-dimensional rotating fixing support and a fixing bottom support; the fixed base is provided with a fixed pin and an azimuth scale; the three-dimensional rotating fixing support is connected with the fixing pin through a cylindrical opening at the bottom, can rotate around the fixing pin in the direction and is locked and fixed; the rotation degree of the three-dimensional rotating fixed support can be read through the azimuth scale; the bottom of the head fixing mechanism is connected with the top of the three-dimensional rotating fixing support, and can perform pitching rotation and locking fixation relative to the three-dimensional rotating fixing support; the rotation degree of the head fixing mechanism can be read through the pitching scale on the three-dimensional rotating fixing support; the two ear rods and the tooth supports are arranged on the head fixing mechanism; when the animal ear support is used, one end parts of the two ear rods can abut against external ear holes of two ears of an animal and are locked and fixed, and the tooth support can be locked and fixed after being placed in an animal mouth and abutting against the upper incisor.
When in use, the two ear rods and the tooth supports are taken down firstly, so that the anesthetized animal lies on the fixed bottom support, and the head of the anesthetized animal is placed in the middle of the head fixing mechanism; then the two ear rods are respectively placed into the left side frame and the right side frame of the head fixing mechanism, and the conical ends of the two ear rods are tightly propped against the external ear holes of the two ears of the animal and then are locked and fixed. The tooth support bracket penetrates into a right side frame of the head fixing mechanism, and meanwhile, the tooth support is placed into an animal mouth and is locked and fixed after abutting against the upper incisor. Controlling the head fixing mechanism to rotate in azimuth and pitch to align the eyes of the animal to the testing lens of the inspection instrument, locking and fixing the head fixing mechanism, and reading and recording azimuth rotation degree and pitch rotation degree of the head fixing mechanism through azimuth scale and pitch scale; the horizontal positions of the two ear rods are read out through the horizontal scales and recorded, so that the accurate adjustment and three-dimensional positioning of the eyes of the animal can be realized.
The invention reflects the influence of different illumination light sources on the development of the eye axis by detecting the length change of the eye axis of the monkey bred in the standard environment. Artificial lighting sources have many features, mainly: spectral distribution, color temperature, brightness, color rendering index, stroboscopic index, etc. Different combinations of these features are key to distinguishing one light source from another. Since the eye is the organ that senses light and the development process is regulated by the illumination in the environment, it is necessary to detect which light source has a regulating effect on the development of the eyeball, especially the growth of the axis of the eye.
The invention utilizes experimental animals of macaca (such as rhesus macaque, cynomolgus monkey, etc.) which are close to the human visual system to research the influence of artificial lighting light on the development of the ocular axis and myopia. In the experimental operation process, concave lenses (negative lenses) or diffusion lenses (diffusing lenses) are not specially arranged outside the eyes of experimental animals, and the animals are not harmfully treated in the experimental process, so that the method is a research method which is closer to the development state of natural myopia.
The head of the experimental animal is fixed by using the positioning instrument when the visual indexes of the experimental animal are detected, the positioning instrument is simple in structure, convenient to use and accurate in positioning, interference caused by change of the position of eyes can be reduced, and stability of multiple times of inspection data of the same animal is achieved.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Fig. 1 is a schematic structural diagram of an animal ophthalmic examination stereotaxic apparatus of the present invention.
Fig. 2 is a side view of the present invention.
Fig. 3 is a schematic view of the fixing plate of the present invention.
In the figure: 1. head fixing mechanism, 11. bottom frame; 12. a left side frame; 13. a right side frame; 2. a three-dimensional rotating fixed support; 21. a circular hole; 22. pitching scales; 23. cylindrical opening; 24. 25, bolts; 3. fixing the bottom support; 31. a fixing pin; 32. azimuth scales; 41. an ear stem; 411. horizontal calibration; 42. a bolt; 43. an ear stem; 431. horizontal calibration; 44. a bolt; 45. a tooth support; 46. a tooth support bracket; 47. and (4) bolts.
Detailed Description
Taking macaque as an example, the method for the comparative study of the illumination light source on the development of the axis of eyes of the macaque animal comprises the following steps:
firstly, selecting healthy male macaques with the age of about 24 months to carry out vision development state detection, screening 32 macaque animals with visual indexes meeting experimental requirements from the healthy male macaques as experimental animals, and simultaneously recording the screening detection values of the visual indexes of the experimental animals, including screening the length of an eye axisDetection value SALiEye diopter screening test value SDiAnterior chamber depth screening test value SCDiCorneal curvature screening test value SKi(ii) a After statistical analysis, when the p value between the standard value and the eye axis length screening detection value, the eye diopter screening detection value, the anterior chamber depth screening detection value, the cornea curvature screening detection value and the like is more than or equal to 0.05, the experiment requirements are considered to be met; wherein i is the experimental animal number.
And secondly, randomly dividing 32 macaques into 4 groups, wherein each group comprises 8 macaques, and at the moment, the interference factor can be controlled to be the lowest. Then, 4 groups of macaques are respectively placed in 4 feeding cages, and the 4 feeding cages are respectively placed in 4 feeding rooms to feed the experimental animals; one of the feeding rooms is illuminated by natural light, and the other 3 feeding rooms are respectively illuminated by an LED lamp, a fluorescent lamp and an incandescent lamp. The illumination of the center position in the rearing cage is 200 and 750lx, the temperature in the rearing room is controlled between 20 and 25 ℃, the relative humidity is controlled between 40 and 70 percent, and the ventilation frequency is not lower than 8 times/hour. The size of the breeding room is 5m × 3m × 2.6 m. The four walls and the ceiling of the room are made of purified color steel plates, and the floor is a light gray epoxy floor paint floor. The activity space in the rearing cage is 80cm multiplied by 80cm, the total height is 140cm, and the material is 304 matte stainless steel.
Third, the experimental animals usually generate stress responses under new illumination conditions, and the stress responses of different experimental animals have certain differences according to different adaptive capacities to new environments, so that the accuracy of experimental results is influenced; therefore, after the test animals are raised for one week, the vision development state of the eyes of each test animal is detected again to obtain the initial detection value of the visual index of each test animal at the moment; including an initial measurement value S of the length of the eye axisALi' eye diopter initial detection value SDi', initial anterior chamber depth measurement value SCDi', initial corneal curvature measurement value SKi'; excluding experimental animals of which the initial detection values are obviously different from the corresponding screening detection values, taking the average value of the initial detection values and the screening detection values of the rest of the experimental animals as a basic detection value, and continuously feeding; the basic detection value comprises an eye axis length basic detection value ZALiEye, eyeDiopter basic detection value ZDiBasal anterior chamber depth measurement ZCDiCorneal curvature basic measurement value ZKi(ii) a It is generally considered that the initial value S of the length of the eye axis is measured after statistical analysisALi' and screening test value SALiInitial detection value Z of interocular diopterDiAnd screening the detected value SDiInitial measurement value S of anterior chamber and anterior chamber depthCDi' and screening test value SCDiInter-or corneal curvature initial measurement value SKi' and screening test value SKiWhen the p value therebetween is less than 0.05, a significant difference between the two is considered.
Step four, after the animals are raised for 6 months, detecting the visual development state of the eyes of each experimental animal again to obtain the final detection value of the visual index of each experimental animal; including the basic detection value Z of the length of the eye axisALI' eye diopter basic detection value ZDI', anterior chamber depth basic detection value ZCDI', corneal curvature basic measurement value ZKI'; in the feeding process, the spectrum of the illumination light source and the illumination in the feeding cage are regularly detected to ensure the stability of the spectrum and the illumination condition in the experimental process; meanwhile, the lighting source replaced in the experiment is reserved regularly and is used as a light source sample to be stored, after the experiment is finished, optical parameters such as spectrum and brightness of the experiment light source are measured again, and consistency of the lighting parameters used in the experiment is guaranteed.
And fifthly, carrying out comparative analysis according to the basic detection value and the final detection value of the visual index of each group of experimental animals to obtain analysis data of the influence of different illumination light sources on the visual development of the experimental animals.
When the visual index of the macaque is detected, the macaque is subjected to conventional anesthesia, and after the macaque does not have pain sensation reaction, the head of the macaque is fixed on a positioning instrument and starts to be detected facing a detection instrument. If the two eyes of the macaque are closed in the detection process, the eyelid opening device is used for opening the upper eyelid and the lower eyelid of the macaque, then the examination of various indexes is carried out, and the artificial tears are regularly dropped on the surfaces of the eyes of the macaque in the examination process, so that the surface of the eyeball of the macaque is ensured to be wet.
As shown, the locator
The lighting parameters of the lighting sources of each rearing cage are shown in the table 1.
TABLE 1
The illumination light source can also be a halogen lamp, an electrodeless sulfur lamp and the like.
As shown in fig. 1, 2 and 3, the positioning apparatus includes a head fixing mechanism 1, a three-dimensional rotating fixing bracket 2, and a fixing base 3.
The fixing base 3 is provided with a fixing pin 31 and an orientation scale 31.
The three-dimensional rotating fixing support 2 is connected with a fixing pin 31 through a cylindrical opening 23 at the bottom, a bolt 25 is positioned in a screw hole on the three-dimensional rotating fixing support 2, and the three-dimensional rotating fixing support 2 and the fixing bottom support 3 can be locked and fixed through the bolt 25; after the bolt 25 is loosened, the three-dimensional rotating fixing bracket 2 can rotate around the fixing pin 31 in an azimuth direction, and the degree of rotation can be read through the azimuth scale 31.
The head fixing mechanism 1 can adopt a rectangular frame; the bottom frame 11 of the head fixing mechanism 1 is used as a pitch axis and is penetrated by a round hole 21 at the top of the three-dimensional rotating fixing support 2; the bolt 24 is arranged in a screw hole at the upper part of the three-dimensional rotating fixing support 2 and can lock and fix the head fixing mechanism 1. When the bolt 24 is loosened, the head fixing mechanism 1 can realize pitching rotation, and the rotation angle can be read through the pitching scale 22 outside the round hole 21.
Two ear rods 41 and 43 with square sections are respectively penetrated through square holes on the left side frame 12 and the right side frame 13 of the head fixing mechanism 1 and are locked and fixed through bolts 42 and 44 arranged in threaded holes of the left side frame 12 and the right side frame 13; the end parts of the two ear rods 41 and 43 which are pressed against the external ear holes of the ears of the animals are conical. When the bolts 42 and 44 are loosened, the two ear rods 41 and 43 can move left and right; the tooth support bracket 46 is composed of two sections of straight rods which are perpendicular to each other, the tooth support 45 is fixed at the end part of one section of the straight rod and faces the rear side, the tooth support can be placed in an animal mouth to tightly abut against the upper incisor, and the other section of the straight rod penetrates through a square hole of the right side frame 13 of the head fixing mechanism 1 and is fixed by a bolt 47.
The head fixing mechanism 1 can also adopt other shapes capable of fixing the head of an animal; the two ear rods 41 and 43 can also be connected with the head fixing mechanism 1 in other modes, as long as the head fixing mechanism 1 can horizontally move left and right and is locked and fixed; the tooth support bracket 46 can also be connected to the bottom frame 11, as long as the tooth support 45 can be moved back and forth, placed in the animal mouth, abutted against the upper incisor and then locked and fixed.
Claims (6)
1. A method for researching the comparison of the development of the eye axis of an animal of the genus Macaca by an illuminating light source is characterized by comprising the following steps:
selecting a plurality of healthy macaca animals with ages of 3-24 months, detecting the visual development state of the healthy macaca animals, screening the macaca animals with visual indexes meeting the experimental requirements as experimental animals, and simultaneously recording the visual index screening detection values S of the experimental animalsiI is the number of the experimental animal;
step two, randomly dividing the screened experimental animals into N groups, and respectively placing the N groups of experimental animals in N rearing cages, wherein N is more than or equal to 2, and the number of the experimental animals in each group is not less than 6; then respectively placing N-1 rearing cages in rearing rooms with different illumination light sources to breed the experimental animals; one breeding room is illuminated by natural light;
step three, after raising for one week, detecting the visual development state of the eyes of each experimental animal to obtain the initial visual index detection value S of each experimental animal at the momenti'; excluding the initial detection value Si' and screening test value SiThe experimental animals with obvious difference are subjected to initial detection values S of the other experimental animalsi' and screening test value SiTaking the average value as the basic detection value Z of the visual index of the experimental animal with the number iiAnd continuing to raise;
step four, after raising for 6-24 months, detecting the vision development state of each experimental animal eye again to obtain each fruitVisual index final detection value Z of animal testi‘；
Step five, according to the basic detection value Z of the visual index of each experimental animaliAnd the final detection value Zi' comparative analysis is carried out, thereby obtaining the analysis data of the influence of different illumination light sources on the visual development of the experimental animal.
2. The method of claim 1, wherein the illumination light source comprises two or more of LED, fluorescent, incandescent, halogen, and electrodeless sulfur lamps.
3. The method as claimed in claim 1, wherein the illumination light source has an illuminance of 200-750lx at the center of the cage.
4. The method of claim 1, wherein the temperature in the breeding room is controlled to be 20-25 deg.C, the relative humidity is controlled to be 40-70%, and the air exchange frequency is not less than 8 times/hr.
5. The method of claim 1, wherein the visual indicator is primarily eye length.
6. The method for researching the contrast of the illuminating light source to the development of the eye axis of the macaque animal according to claim 1, wherein in the third step and the fourth step, the spectrum of the illuminating light source and the illumination in the rearing cage are regularly detected, so that the stability of the spectrum and the illumination in the experimental process is ensured; meanwhile, the lighting source replaced in the experiment is reserved as a light source sample for storage at regular intervals.
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| CN201711273533.7A CN107981837B (en) | 2017-12-06 | 2017-12-06 | Comparative research method for yangtao axis development of macaca by using illumination light source |
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| CN102884370A (en) * | 2010-03-17 | 2013-01-16 | 万斯创新公司 | Light sources adapted to spectral sensitivity of diurnal avians and humans |
| CN103598143A (en) * | 2013-11-18 | 2014-02-26 | 浙江大学 | Mouse myopia model, and inducing device and inducing method thereof |
| CN104540297A (en) * | 2015-01-08 | 2015-04-22 | 复旦大学附属金山医院 | Stroboscopic dimming device for animal experiments |
| CN107049228A (en) * | 2016-12-31 | 2017-08-18 | 上海复展智能科技股份有限公司 | The adjustable vision testing system of illumination spectrum |
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| CN101828529A (en) * | 2010-01-29 | 2010-09-15 | 中国科学院武汉物理与数学研究所 | Rat dual-visual cue-training and detecting experimental system |
| CN102884370A (en) * | 2010-03-17 | 2013-01-16 | 万斯创新公司 | Light sources adapted to spectral sensitivity of diurnal avians and humans |
| CN103598143A (en) * | 2013-11-18 | 2014-02-26 | 浙江大学 | Mouse myopia model, and inducing device and inducing method thereof |
| CN104540297A (en) * | 2015-01-08 | 2015-04-22 | 复旦大学附属金山医院 | Stroboscopic dimming device for animal experiments |
| CN107049228A (en) * | 2016-12-31 | 2017-08-18 | 上海复展智能科技股份有限公司 | The adjustable vision testing system of illumination spectrum |
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