CN113133431A - Establishment method, model and application of chronic ocular hypertension combined long-axis animal model - Google Patents
Establishment method, model and application of chronic ocular hypertension combined long-axis animal model Download PDFInfo
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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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
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Abstract
The invention discloses a method for establishing an animal model with chronic ocular hypertension combined with a long axis of eyes, which comprises the following steps: s10, anaesthetizing the model animal; s20, intermittently suturing the conjunctiva and the superficial sclera for a circle by using a non-absorbable suture line along the periphery of the cornea at a set distance; s30, tightening the suture, and knotting and fixing; and S40, measuring the intraocular pressure of the model animal within 5 minutes after knotting, and if the intraocular pressure is more than 60mmHg (1mmHg is 0.133kPa), obtaining the model of the animal with chronic ocular hypertension and long axis of eyes. Can increase the intraocular pressure of model animals and increase the ocular axis, and has the advantages of long intraocular pressure maintaining time, less ocular complications, simple operation and high success rate of the operation. In addition, the invention also discloses an animal model with chronic ocular hypertension combined with a long axis of eyes and application thereof in researching glaucoma combined with myopia.
Description
Technical Field
The invention relates to an animal disease model, in particular to a method for establishing an animal model with chronic ocular hypertension and long ocular axis. The invention also relates to a chronic ocular hypertension combined long-axis animal model and application of the chronic ocular hypertension combined long-axis animal model in experimental research on glaucoma combined myopia diseases.
Background
Glaucoma is a type of irreversible blinding eye disease mainly manifested by optic nerve damage and visual field defect, and is essentially irreversible death of Retinal Ganglion Cells (RGCs), and the main risk factor is pathological Intraocular Pressure (IOP) elevation. Currently, the primary treatment regimen for glaucoma is lowering IOP by medication or surgical methods. The main clinical characteristics of pathological myopia are that the diopter is larger than-6D, the ocular axis is progressively lengthened, and the retinal pigment epithelium and choroid are thinned in the ocular fundus. In clinical work, the incidence of myopia is high, the prognosis of glaucoma treatment is poor, and patients with common open-angle glaucoma complicated with high myopia are difficult to distinguish and diagnose the damage to optic nerves and the peripheral damage of the optic nerves and the characteristic optic nerve change of glaucoma, so that an appropriate animal model is needed as a research basis in the mechanism research of two complicated diseases and the aspect of optic nerve protection treatment.
At present, the common operation construction method and the characteristics of the chronic ocular hypertension animal model of glaucoma are as follows: (1) anterior chamber injection blocking trabecular meshwork methods such as injection of microbeads, chemically cross-linked hydrogels, chondroitin sulfate, carbomer, sodium hyaluronate, methyl cellulose, etc., the anterior chamber injection method having: the intraocular pressure is obviously increased, but the maintenance time of the injection, particularly sodium hyaluronate, carbomer and the like is short, the injection is possibly required to be repeatedly injected, the operation relates to intraocular injection, the intraocular inflammatory reaction is possibly caused, and the toxic effect of the injection on retina is possibly interfered with the test result; (2) laser photocoagulation of trabecular meshwork, pericorneal limbal vascular meshwork, scleral blood vessels, etc., the laser photocoagulation method has: the requirement on surgical equipment is high, and the success rate is related to the laser intensity, the repeated photocoagulation times and the like; (3) drug induction method: such as corticosteroid hormone, tumor necrosis factor beta (TGF-beta), etc. injected into the vitreous cavity,such methods have: the injection in the vitreous cavity causes retina toxic reaction, and intraocular operation has the characteristic of causing endophthalmitis risk; (4) transgenic animals: such as GpnmbR150X and Tyrp1isaRelated DBA/2J mice, and Tyr437Related Tg-MYOC mice, Sh3pxd2b related B6.Sh3pxd2bneeMice, etc., also characterized by chronic ocular hypertension, such animals having: the model construction period is long, and the economic cost is high. The existing glaucoma high-intraocular-pressure animal model mostly focuses on simulation and research of intraocular pressure increase on retinal damage and retinal ganglion cell number and function change, and a simple and effective animal model which can be applied to glaucoma and myopia is not available.
Based on the clinical diagnosis and treatment difficulty of glaucoma, especially open-angle glaucoma complicated with high myopia, it is urgently needed to establish a long-term effective chronic ocular hypertension complicated with long axis of the eye animal model, which has important significance for researching the pathogenesis of glaucoma complicated with pathological myopia and searching new treatment methods and medicines for preventing, treating or delaying the course of glaucoma and pathological myopia.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for establishing a model of chronic ocular hypertension combined with a long axis animal, which can be used for establishing the model of chronic ocular hypertension combined with the long axis animal by an operation, and has low establishing cost and high stability.
The invention further aims to solve the technical problem of providing an animal model combining chronic ocular hypertension and long ocular axis, which is convenient to manufacture and high in model stability.
The invention also aims to solve the technical problem of providing an application of an animal model with chronic ocular hypertension and long ocular axis in experimental research on glaucoma and myopia complicated diseases.
In order to achieve the above object, the present invention provides, in one aspect, a method for establishing an animal model of chronic ocular hypertension combined with a long axis of the eye, comprising the steps of: s10, anaesthetizing the model animal; s20, intermittently suturing the conjunctiva and the superficial sclera for a circle by using a non-absorbable suture line along the periphery of the cornea at a set distance; s30, tightening the suture, and knotting and fixing; and S40, measuring the intraocular pressure of the model animal within 5 minutes after knotting, and if the intraocular pressure is more than 60mmHg, obtaining the model of the animal with chronic ocular hypertension and long axis of eyes.
Preferably, in step S20, the set distance is 2.5-3 mm.
Further preferably, in step S20, the intermittent sewing gauge is 3-4 mm.
Preferably, in step S30, the amount of cinching of the cinched suture is 4-6 mm.
As a preferred method, in step S10, the method of anesthesia includes topical anesthesia.
Specifically, the surface anesthetic is dripped into eyes of model animals in two times, 1-3 drops are dripped each time, and the interval time between two dripping is 8-10 minutes.
Preferably, the eyelid is distracted using an eyelid distractor with a positioning function, and the suture position and the suture gauge are measured by the eyelid distractor.
Preferably, in step S20, the non-absorbable suture is 7-0 suture; in step S30, the knot is fixed by three-fold knot.
According to a second aspect of the invention, a model of chronic ocular hypertension complicated with a longaxis oculomotor is provided, which is manufactured according to any one of the methods for establishing the model of chronic ocular hypertension complicated with a longaxis oculomotor provided by the first aspect of the invention.
In a third aspect, the invention provides an application of an animal model with chronic ocular hypertension and long ocular axis in researching glaucoma and myopia complicated diseases.
Compared with the prior art, the model of the animal model with the chronic ocular hypertension and the long axis of the eyes has the advantages that after suture is tightened, the tension of the corneosclera edge of the model animal is increased, the backflow of aqueous humor is blocked, and the long-term chronic increase of the anterior chamber pressure is caused. Compared with other intraocular injection constructed ocular hypertension models, the ocular hypertension model does not penetrate the anterior chamber and does not generate inflammatory reaction of the anterior chamber and retina directly caused by intraocular injection. In addition, the suture line around the corneal limbus can cause mechanical/ischemic mixed factors due to mechanical compression of the suture line, so that the sclera is anoxic, and further, the sclera is remodeled to cause the growth of the ocular axis, thereby being suitable for pressure optic nerve damage. The model establishes that the early transient ocular hypertension can simulate the acute attack stage of the primary angle-closure glaucoma, and the later chronic mild increased ocular tension simulates the change of the ocular tension after the attack, so that the process of the chronic glaucoma causes the increase of the ocular axis while the ocular tension is increased, and does not generate large complications.
Drawings
FIG. 1 is a flow chart of one embodiment of a method for modeling an animal with chronic ocular hypertension combined with a long axis of the eye according to the present invention;
FIG. 2 is a schematic view of a positionable eyelid distractor used in embodiments of the present invention;
FIG. 3 is a schematic view of a suture site of an eyeball in one embodiment of the invention;
FIG. 4 is a graph of the change in rat cornea before (A) and after (B) knotting sutures in one embodiment of the present invention;
FIG. 5 is a graph showing intraocular pressure measurements taken at various time periods before and after a rat circumcorneal limbus suture in accordance with one embodiment of the present invention;
FIG. 6 is a graph of HE staining of the corneal limbus of a rat 4 weeks after surgery in accordance with an embodiment of the present invention;
FIG. 7 is an enlarged view of a portion A of FIG. 6;
FIG. 8 is a partial enlarged view of portion B of FIG. 6;
FIG. 9 is a graph of HE staining of rat retinas at 4 weeks post-surgery in accordance with an embodiment of the invention;
FIG. 10 is a graph of ganglion cell counts at various sites in the retina of a rat 4 weeks after surgery, according to an embodiment of the invention;
FIG. 11 is a graph of statistical analysis of ganglion cell counts at different sites in rat retina 4 weeks after surgery in accordance with an embodiment of the present invention;
FIG. 12 is a transmission electron microscope image of the sclera of a rat 4 weeks after surgery in accordance with an embodiment of the present invention;
FIG. 13 is a graph of statistical analysis of the results of post-operative 4-week rat ocular axis measurements in accordance with an embodiment of the present invention;
FIG. 14 is a MASSON staining pattern of rat sclera at 4 weeks post-surgery in accordance with an embodiment of the present invention;
FIG. 15 is a schematic diagram of a semi-quantitative analysis of collagen volume fraction in the posterior segment of rat eyeball at 4 weeks post-operation in accordance with one embodiment of the present invention;
FIG. 16 is an electrogram of a rat retina 4 weeks after surgery in one embodiment of the invention;
FIG. 17 is a graph of visual evoked potentials of rats 4 weeks after surgery in accordance with an embodiment of the present invention;
FIG. 18 is a graph of rat sclerostin α -SMA expression at 6 months post-surgery in one embodiment of the invention;
FIG. 19 is a graph of a 6 month post-operative quantitative analysis of rat sclera immunoblot grayscale in accordance with an embodiment of the present invention.
Description of the reference numerals
11 superior rectus muscle and 12 superior oblique muscle
13 inner rectus muscle 14 inferior oblique muscle
15 lower rectus muscle 16 outer rectus muscle
2 episcleral vein 3 cornea
4 sclera 5 suture
51 three-fold knot
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The present invention is described in detail below by way of examples, and it is to be understood that the embodiments described herein are merely illustrative and explanatory of the present invention, and the scope of the present invention is not limited to the embodiments described below.
In a specific embodiment of the invention:
the experimental animal SD rat used was purchased from Silikzeda laboratory animals Co., Ltd, Hunan province (certification number: 430727201101172826).
Drugs and reagents used: α -SMA is available from Abcam corporation, UK (ab 5694); the cytoplasm protein extraction reagent is tissue lysate RIPA (Servicebio, 30ml), protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (Servicebio,100mM/1ml) (RIPA: PMSF ═ 20: 1); BCA protein concentration assay kit (Servicebio, 200T); reduced protein loading buffer (Servicebio,1 ml). Other drugs and agents are conventional commercial products.
The apparatus and instrument used, tonometer is Tonolab type rebound tonometer produced by Vantaa Icare corporation of finland; the ophthalmic A/B type ultrasonic diagnostic apparatus is Tianjin Mada ODM-2100S; the fluorescence microscope is germany LeikaDM 5000B; transmission Electron Microscopy (TEM) is hitachi HT 770; electroretinogram (ERG) Ganzfelz Electroretinogram (stimulator: Q450) manufactured by Rowland, Germany; visual Evoked Potentials (VEPs) were detected using a national medical multifocal Visual electrophysiological system. Others are all common commercial products.
The operation of the method and the effect thereof are further illustrated by the following specific examples.
Example 1:
this example was used to model chronic ocular hypertension with long ocular axis animals.
Preparation of model animals:
40 SD healthy male rats of 7-8 weeks old were selected and randomly divided into two groups of 20 rats each, which were used as a model group and a control group, respectively. Raising in free-feeding drinking water environment at 20-26 deg.C and relative humidity of 60% for one week. Intraocular pressure was measured 3 days before surgery on an awake basis at 8-10 points daily and recorded as baseline intraocular pressure from the average intraocular pressure value 1 day before surgery, after which intraocular pressure measurements were taken at this time interval.
Establishing an animal model:
the process of establishing the animal model is shown in figure 1 and mainly comprises the following steps:
s10, anesthetizing the model animal: after general anesthesia was performed by injecting 2% sodium pentobarbital into the abdominal cavity of rats of the model group and the control group at a dose of 80mg/kg (0.3ml/100g), the skin around the eyes of the rats was sterilized with iodophor, and the eyes were washed with povidone-iodine and physiological saline. An operating sheet is laid on the eyes of the rat, an eyelid spreader is placed on the eyes of the rat to spread the eyelids of the rat, and 2 drops of 0.4% oxybuprocaine hydrochloride eye drops are respectively dripped into the two eyes of the rat to perform surface anesthesia on the ocular surface. Ten minutes later, the eyelid spreader was placed, and 2 drops of oxybuprocaine hydrochloride eye drops were added again for surface anesthesia once.
It should be understood that various practical methods of anesthesia can be used to perform general anesthesia and topical anesthesia on the eyes of rats, which are considered satisfactory as far as the animals have no adverse reaction to the systemic and topical stimulation of the eyes, and the use of a specific anesthetic is not limited in the present invention.
S20, suturing the sclera part of the model animal: after the conjunctiva is taken out by using the microscope forceps without irritation reaction, the anesthesia is considered satisfactory, and at the moment, the eyelid of the rat is spread by using an eyelid spreader. The eyelid distractor may be a self-made positionable eyelid distractor as shown in fig. 2, and the structure and method of use of the positionable eyelid distractor may be found in utility model patent publication No. CN 212307923U. The eyelid spreader can be positioned to help position the suture position, facilitate the suture of the sclera 4 and improve the suture precision and the identity of the eye features of the model animal. Of course, the rat's sclera can also be sutured by spreading the rat's eyelid using a common commercially available eyelid spreader. As shown in a of fig. 3, the sclera 4 outside the cornea 3 of the model group rat was intermittently sutured for one week using a non-absorbable suture No. 7-0 5. The control rats were not sutured.
Specifically, the sclera suture can be carried out by using a No. 3 suture, the space between the suture 5 and the corneoscleral limbus is controlled to be 2.5-3mm, the needle pitch is controlled to be about 4mm, the suture needle penetrates through the conjunctiva and the superficial sclera during the suture, the cornea limbus is sutured 360 degrees, and 5-6 needles are sutured together. The close distance between the suture 5 and the corneoscleral margin is easy to cause ischemia of the blood vessel network of the corneosclera, perforation of corneal ulcer and other adverse effects; too far distance may cause compression of extraocular muscles, scleral ischemia, and gradual posterior displacement of sutures with eye movement, possibly reducing the effects of chronic persistent ocular hypertension model by mechanical stress. As shown in fig. 2, in the sclera part of the rat, there are a plurality of oculomotor muscles such as superior rectus muscle 11, superior oblique muscle 12, internal rectus muscle 13, inferior oblique muscle 14, inferior rectus muscle 15, and external rectus muscle 16, and a plurality of blood vessels such as episcleral vein 2. During suturing, the thick conjunctival blood vessels and episcleral veins 2 are avoided, so that the conjunctival hematoma is prevented from influencing the tightening, pressing and knotting of the suture 5; care was also taken to avoid individual oculomotor muscles so as not to affect the rotation of the rat's eyes. The needle does not penetrate the sclera, thereby avoiding a reduction in basal intraocular pressure and increasing the risk of intraocular inflammation.
And S30, tightening and knotting. The suture is tightened from both ends of the suture 5 sutured on the sclera of the rat, the tightening amount is 4-6mm, and the suture is tied and fixed after being tightened. Insufficient pressure, short intraocular pressure maintaining time and low model effectiveness are easily caused by too small tightening amount; too much tightening can lead to too much pressure, severe eyeball ischemia, corneal ulcer, retinal necrosis and other conditions with increased failure rate of the model. Specifically, when the suture is knotted and ligated, the first knot is rewound twice to increase the friction surface and friction force between the threads, and the second knot is a single knot and is opposite to the first knot in direction, so that a surgical knot is formed between the 1 st knot and the 2 nd knot; the third knot is opposite to the second knot in direction, so that the 2 nd and 3 rd knots are square knots and form a three-fold knot 51 to strengthen the friction force between the ligation threads and prevent the threads from loosening and slipping. Before knotting, the suture 5 is returned to the suture position, and no dragging action is needed when knotting, so as to avoid tension which can drag the conjunctiva to move towards the cornea.
After suturing, before tightening and knotting the suture, the rat eye is shown in fig. 4A, and the suture 5 is intermittently sutured on the sclera 4 around the cornea 3, when the conjunctiva is not hyperemic and the cornea 3 is transparent. After the suture 5 was tightened and knotted, as shown in the graph B in fig. 4, the rat had a whitish limbus, an edema in the cornea 3, a mydriasis, and a disappearance of the light reflex.
The model animal in the present invention is not limited to rat, and other suitable animals such as guinea pig, rabbit, and the like may be used as the model animal in the present invention. The suture used for suturing, the amount of suture cinching and the method of tying the knot may also be chosen differently depending on the anatomical features of the different model animals used,
s40, obtaining a model of chronic ocular hypertension combined with long axis ocular movement:
intraocular pressure of rats is measured by using a rebound tonometer within 5 minutes after suture knotting is completed, and if the intraocular pressure is more than 60mmHg, the intraocular pressure is indicated to be successfully established by combining chronic ocular hypertension and a long axis animal model.
By the model of combining chronic ocular hypertension and long-axis ocular movement established by the embodiment, the ocular hypertension state can be stably maintained for 2 months by a single operation.
Example 2
This example is used to detect ocular hypertension and its induced changes in the tissues of the ball in a model animal.
Intraocular pressure was measured in the awake state of the rats using tonometers at 1 day, 1 week, 2 weeks, 4 weeks, 2 months, and 6 months after the operation of the model group rats, respectively, and changes in intraocular pressure at different time periods were analyzed, and the measurement results are shown in fig. 5. As can be seen from FIG. 5, after the rat is sutured and molded by the circular limbal suture, the intraocular pressure rises sharply when the suture is ligated. At this time, as shown in panel B of FIG. 4, the rat had ischemic blush limbus, corneal edema, and mydriasis. The pressure of the eyes of the rat in a normal waking state is 10-13mmHg, and the intraocular pressure of the normal control group in the embodiment is 12.2 +/-1.5 mmHg after 5 minutes of operation. The peak value of the intraocular pressure measurement result of the model group can reach 98mmHg, and the ocular pressure is reduced to 61.4 +/-10.4 mmHg after 5 minutes of operation. And gradually decreases over time. The intraocular pressure measured 1 day after the operation is 25.1 +/-3.6 mmHg. After the model is successfully established, the rat has heavy ocular surface inflammatory reaction after 1 week of operation, and the ocular surface inflammatory reaction is manifested as conjunctival congestion and corneal edema. Part of the rats had pupillary dilated and had lost light reflection, but no apparent cloudiness was seen in the anterior chamber. From 2 weeks post-surgery, the inflammatory response was significantly reduced. All successfully modeled rats had a bullseye appearance at 2 weeks post-surgery, had prominent eyes, and were able to maintain an intraocular pressure of 25.6 ± 3.3mmHg at 2 months post-surgery, with statistical differences compared to the control group (t 13.398, P < 0.01). It is demonstrated that the method of the present invention can cause chronic ocular hypertension in rats.
After 4 weeks, 3 rats in the model group and 3 rats in the control group are respectively euthanized, the whole eyeballs are taken out, paraffin sections HE staining is carried out on the eyeballs, and the positions of the suture lines 5 at the corneoscleral edges and the changes of the shapes and the quantities of the cell tissues of all layers of the retinas corresponding to the suture lines 5 are observed under an upright microscope. As shown in FIGS. 6 and 7, the arrangement of the tissues at the corneoscleral edge of the rats in the control group was regular, and no abnormal tissue change was observed; as shown in figures 6 and 8, the suture 5 is visible at a position 2mm outside the corneoscleral edge of the model group rat, and the retinal depression of the corresponding part of the suture 5 shows that the suture exerts a compression effect on the retina of the model group rat. As shown in FIG. 9, the retina structure of the control rat was normal, and the ganglion cells were uniformly distributed; model group rats had a reduced number of retinal ganglion cells and a reduced thickness of the retinal whole layer. Indicating that the retina of the model group rat is obviously damaged.
Taking 3 rats of a model group and a control group which are 3 weeks after operation, respectively, performing general anesthesia, respectively using a rat brain stereotaxic apparatus to perform fluorescence gold tracer marking on retinal ganglion cells by a retrotracking method through a superior mound, performing euthanasia on the rats after 1 week, performing retinal plating after heart perfusion, performing developing radiography on the posterior pole part, the middle peripheral part and the peripheral part (about 1/6, 1/2 and 5/6 parts of the radius of the retina respectively) of four quadrants of the plated retina under an upright fluorescence microscope, and counting the marked retinal ganglion cells. As shown in fig. 10, the numbers of retinal ganglion cells were reduced in the posterior pole (panel B in fig. 10), the middle periphery (panel D in fig. 10) and the peripheral portion (panel F in fig. 10) of the retina of the model group rat, compared with the posterior pole (panel a in fig. 10), the middle periphery (panel C in fig. 10) and the peripheral portion (panel E in fig. 10) of the retina of the control group rat. Indicating that the retinal ganglion cells of the rats in the model group are dead and the number of the retinal ganglion cells is reduced. Statistical analysis was performed on the retinal ganglion cell counts and the results are shown in fig. 11. The analysis results suggest: the model group had a reduction in posterior polar, mid-peripheral and peripheral retinal ganglion cells compared to the control group.
Example 3
This example is used to detect changes in the long-eye axis and scleral tissues of model animals.
Taking 3 rats of a model group and a control group 4 weeks after operation, respectively, euthanizing, taking out and marking complete eyeballs, taking the sclera (2 mm behind the limbus) of the equator, fixing the sclera with 2.5% glutaraldehyde stationary liquid, preparing an electron microscope specimen, and observing the ultrastructure of scleral collagen fibers by using a transmission electron microscope. As a result, as shown in FIG. 12, the number of rats in the model group was increased compared to those in the control group due to the structural disorder of collagen fibers.
After general anesthesia was performed on model group rats and control group rats 4 weeks after the operation, ocular axis measurement was performed on the rats using an ophthalmic a/B type ultrasonic diagnostic apparatus, and statistical analysis was performed on the measurement results, the results of which are shown in fig. 13. As can be seen from fig. 13, the ocular axis of the model group rats was increased compared to the control group rats. Illustrating that the use of the method of the present invention can cause the growth of the axis of the eye of a model animal.
Taking 3 rats of the model group and the control group which are 4 weeks after operation, carrying out euthanasia respectively, taking out complete eyeballs, marking, carrying out MASSON staining on the eyeballs, and observing under an upright microscope. As shown in FIG. 14, the range of collagen fiber staining area of the equatorial eyeball (2 mm posterior to the limbus) of the model group rats (panel B in FIG. 14) was larger than that of the control group rats (panel A in FIG. 14). When the volume of collagen fibers in this area was semi-quantitatively analyzed, as shown in fig. 15, collagen fibers, which are important components for scleral remodeling of the eyeball at the equatorial region, were more present in the model group than in the control group.
After general anesthesia is carried out on model group rats and control group rats 4 weeks after operation, the rats are placed in a dark environment to adapt for more than 12 hours in a dark environment, and ERG examination is carried out after mydriasis. As shown in fig. 16, when the a-wave amplitude and the B-wave latency in the dark adaptation 3.0 mode were compared between the model group rats and the control group rats, it was found that the a-wave amplitude and the B-wave latency of the model group rats (a waveform in fig. 16) were decreased and the B-wave latency was increased compared with the a-wave amplitude of the control group rats (B waveform in fig. 16). Indicating that the function of the retinal photoreceptor cells of the rats in the model group is reduced. The examination of normal pupil size, non-mydriatic rats by flash VEP after general anesthesia, is shown in fig. 17. As can be seen from fig. 17, the P2 wave latency of the VEP white light 3.0 mode of the model group rats (D waveform in fig. 17) was extended compared to the P2 wave latency corresponding to the control group rats (C waveform in fig. 17). Indicating that the retinal ganglion cell-related visual conduction function of the rats in the model group is impaired.
At 6 months after the operation, 3 rats in the model group and 3 rats in the control group are respectively taken, euthanasia is carried out, complete eyeballs are taken out, and fresh scleral tissues at different parts are separated. Appropriate amounts of the cytosolic protein extraction reagents A and B were mixed at a ratio of 20:1 (e.g., 200. mu.l of cytosolic protein extraction reagent A was added with 10. mu.l of extraction reagent B), and phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1mM to prepare a lysate. Lysate was added at a rate of 200. mu.l lysate per 60mg scleral tissue to obtain a tissue homogenate. Performing ice bath, homogenizing at low temperature, centrifuging at 12000rpm for 5min at 4 deg.C, collecting supernatant as protein solution, and measuring protein concentration with BCA protein concentration measuring kit. Adding the protein solution into 5-reduced protein loading buffer solution according to the ratio of 4:1, performing boiling water bath denaturation for 15min, and storing in a refrigerator at-20 ℃ for later use; SDS-PAGE electrophoresis: cleaning a glass plate, preparing and loading glue, concentrating glue voltage to 75V, carrying out immunoreaction (the dilution concentration of primary anti-alpha-SMA is 1: 3000, the dilution concentration of secondary antibody is 1:5000) after separating glue is subjected to constant-current wet-transfer membrane for half an hour, carrying out chemiluminescence development and fixation, and carrying out gel image analysis. Gel image results are shown in FIG. 18, where the expression level of α -SMA protein in the sclera of the model group rats was higher than that of the control group rats. The results of the quantitative grayscale analysis of immunoblots are shown in FIG. 19, where the model rats had significantly increased α -SMA content in their sclera compared to the sclera at different sites in the control group. The surgical method used by the model group is characterized in that the pressure source is mainly suture compression blocking of an aqueous outflow channel, scleral mechanical stress conduction is proved to be the main reason of scleral remodeling during the development of myopia and glaucoma diseases, the stress of an intracellular framework protein (such as alpha-SMA) network is rapidly and directly conducted to extracellular matrix, the asymmetric change of optic nerve papilla is caused, and the like, the result indicates that in glaucoma and myopia diseases, particularly in patients with open-angle glaucoma and high myopia, the alpha-SMA can be used as a regulatory factor to participate in the pathogenesis of non-genetic factors of the patients.
The animal model with chronic ocular hypertension and long ocular axis obtained by the method can be widely applied to the research of glaucoma and myopia.
In conclusion, in the embodiment of the method for establishing the animal model with chronic ocular hypertension and long ocular axis, the 7-0 non-absorbable suture line is annularly and discontinuously sewed at the position 2.5-3mm away from the corneal limbus, so that the time for maintaining the ocular hypertension is remarkably increased, and the ocular axis of the animal model is in a growth state while the ocular hypertension is increased, and no other ophthalmic complications are generated. The chronic ocular hypertension combined long-axis ocular hypertension animal model established by the method can stably keep the ocular hypertension above 25mmHg, the modeling success rate is above 90%, the ocular hypertension stable maintenance time is above 4 weeks, the surgical wound is small, the inflammatory reaction of eyes is light, the cornea has no obvious edema and new vessels, the modeling operation is simple, and the surgical operation can be completed within 10 minutes.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A method for establishing an animal model with chronic ocular hypertension and long ocular axis is characterized by comprising the following steps:
s10, anaesthetizing the model animal;
s20, intermittently suturing the conjunctiva and the superficial sclera for a circle by using a non-absorbable suture line along the periphery of the cornea at a set distance;
s30, tightening the suture, and knotting and fixing;
and S40, measuring the intraocular pressure of the model animal within 5 minutes after knotting, and if the intraocular pressure is more than 60mmHg, obtaining the model of the animal with chronic ocular hypertension and long axis of eyes.
2. The method according to claim 1, wherein in step S20, the set distance is 2.5-3 mm.
3. The method of claim 2, wherein in step S20, the intermittent stitching has a stitch gauge of 3-4 mm.
4. The method of claim 1, wherein in step S30, the amount of cinching of the cinched suture is 4-6 mm.
5. The method according to any one of claims 1 to 4, wherein in step S10, the method of anaesthesia comprises topical anaesthesia.
6. The method of claim 5, wherein the topical anesthetic is applied to the eye of the model animal in two drops, 1-3 drops each, with an interval of 8-10 minutes.
7. The method according to any one of claims 1 to 4, wherein the eyelid is distracted using an eyelid distractor with a positioning function, and the suture position and the suture gauge are measured by the eyelid distractor.
8. The method according to any one of claims 1 to 4, wherein in step S30, the method of knot fixing is three-fold knot fixing.
9. An animal model of chronic ocular hypertension incorporating the long axis of the eye, made according to the method of any one of claims 1 to 8.
10. Application of an animal model with chronic ocular hypertension and long axis of eyes in researching glaucoma and myopia.
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