CN112568187A - Method for constructing novel mouse model with retinal vein occlusion - Google Patents
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Abstract
The invention relates to the technical field of biomedicine, in particular to a method for constructing a novel mouse model for retinal vein occlusion. The method for constructing the novel mouse model with retinal vein occlusion comprises the step of interfering the injection of the Muller cell metabolism inhibitor into the posterior vitreous body of the mouse by adopting a photodynamic method. The mouse model for retinal vein occlusion prepared by the construction method is stable, can realize persistent retinal edema, is convenient for verifying drug targets and action mechanisms of candidate drugs after injection, and provides a new generation of animal model tools for the research of retinal vein occlusion diseases.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for constructing a novel mouse model with retinal vein occlusion.
Background
Retinal Vein Occlusion (RVO) is one of the common retinal vascular diseases in ophthalmology, the incidence rate of which is second only to diabetic retinopathy, and about 1430 million adults are affected worldwide. RVOs can be classified into Branch Retinal Vein Occlusion (BRVO), Central Retinal Vein Occlusion (CRVO), and hemilateral retinal vein occlusion (HCRVO) according to the location of occlusion. RVO is usually caused by thrombosis, and as the disease progresses, complications such as Macular Edema (ME) and neovascular glaucoma secondary to RVO may result, causing severe visual deterioration and even blindness. Currently, methods of ME treatment secondary to RVO include macular grid laser photocoagulation, intraocular injection of long-acting hormone-like drugs, and intraocular injection of anti-Vascular Endothelial Growth Factor (VEGF) drugs, among others. Wherein, because the intraocular injection of the anti-VEGF treatment RVO has better curative effect on the improvement of the vision of patients, has little side effect and is widely accepted by clinicians and patients. First line of China anti-VEGF drugs for ME secondary to RVO (macular edema) include Cupressixi generalized ranibizumab (lucentis), both of which exert effects of treating ME progression by specifically antagonizing VEGF. The previous experimental research shows that the ranibizumab mainly recognizes the human VEGF, and the recognition efficiency of the ranibizumab on the mouse VEGF is low. Thus, combo-xipu is more suitable for the novel RVO mouse model validation.
Animals currently used for RVO model construction include rodents (mice and rats), cats, dogs, pigs, and non-human primates, all of which have their own advantages and disadvantages. While small animals have relatively small eyeballs but are convenient to feed and relatively inexpensive, large animals have large eyeballs but are expensive and difficult to feed and manipulate, model animals other than human primates do not have a macular structure and thus can only simulate ME by inducing retinal edema. In view of economic cost, operational feasibility and ethical issues, mice are the most commonly used animal for RVO model construction. The RVO construction method comprises a blood vessel ligation method, a method for injecting endothelin-1 into a vitreous cavity, a laser photocoagulation retinal vein sealing method and a photodynamic method. The experimental RVO animal model manufactured by the method is proved to have a series of typical RVO expression and pathological characteristics such as hemorrhage, edema, blood flow stasis, microvascular dilatation leakage and the like on the retina after 24h of RVO molding through fundus photography, FFA and histological examination, but each method has respective advantages and disadvantages, particularly in the aspects of operability of the manufacturing method, traumatism to experimental animals, recanalization time of blocked blood vessels and the like. The periorbital lateral wall needs to be cut open by the vascular ligation method, the operative wound is large, the infection chance of the animal after the operation is increased, the survival rate is obviously reduced, and the long-term observation of the experimental result is seriously influenced. The method of injecting endothelin-1 into vitreous cavity to induce RVO is caused by severe spasm of blood vessel, has a great difference with human RVO forming mechanism, is only suitable for researching electrophysiological change after retinal ischemia, and is not suitable for being used as experimental treatment model. The blood vessel blocked by the laser photocoagulation retinal vein occlusion method is re-passed for 3 days, repeated photocoagulation is needed for many times, and serious thermal damage to retinal pigment epithelium, photoreceptor cells and retinal inner tissue can be caused. More importantly, laser photocoagulation is a laser to coagulate blood rather than to form a thrombus, unlike the clinical RVO formation mechanism. The photodynamic method is that after the photosensitive medicine is injected into vein, laser irradiates the pre-blocked retina blood vessel, and the RVO model is formed under the combined action of the photosensitive medicine and the irradiated laser. Compared with other modeling methods, the photodynamic method has the advantages of noninvasive operation and capability of avoiding excessive damage of retinal tissues, but the blocked blood vessels are usually re-introduced about 1 week after modeling. By combining the current RVO animal model situation, the mouse RVO model constructed by the photodynamic method is the most suitable scheme, and the method is also applied to the function verification of a plurality of preclinical medicaments. Photosensitizers currently used in the construction of RVO animal models include bengal and erythrosin B, with bengal being more common. However, further analysis shows that the retinal thickness of the mice in the anti-VEGF group and the control group at the 7 th day after the model making is lower than that of the mice before the model making intervention, and possible reasons for the phenomenon include that blocked blood vessels are re-passed in one week, so that the local edema condition is relieved, and meanwhile, ischemia and hypoxia stimulation caused by the model making causes the increase of apoptosis, the reduction of cell content and the reduction of retinal thickness. The retinal thickness of RVO mice after model building is lower than the baseline level after 1 week, and the target point and candidate drugs to be verified can not play a role until 1 week after intravitreal injection, which causes troubles to related researches, so that a more durable retinal edema model is needed.
Research on the pathogenic mechanism of ME shows that increased leakage and blocked fluid backflow caused by abnormal blood vessels are the causes of retinal edema, but the current technical means that vascular ligation and repeated laser sealing of retinal veins can cause more permanent vascular obstruction, but the vascular ligation and the repeated laser sealing have obvious difference from clinical RVO and large operation damage, and the model making failure is easily caused, so that the feasibility for reducing the inter-retinal tissue fluid backflow is realized. The normal retinal tissue Muller glial cells function as water pumps to maintain a highly ordered layered structure of the retina by pumping out fluid from the inner retina. Dysregulation of Muller glial cell function will significantly increase retinal edema, leading to cystoid edema in the macular region. Therefore, Muller cell function decompensation is a key factor for ME. DL-alpha-aminohexanoic acid (DL-alpha-aminoadipopic, DL-AAA) is a targeted Muller cell metabolism specific inhibitor. The normal function of the Muller cells is specifically inhibited by injecting DL-AAA into the vitreous body, and the structural and functional changes of the retina after the function of the Muller cells is abnormal are further researched. Although there have been subjects reporting leakage of rabbit retina after intravitreal injection of DL-AAA, it is only the leakage that is significantly different from the clinical manifestations of RVO, and there is currently no systematic study on the effects of DL-AAA in causing retinal edema in mice.
Disclosure of Invention
The invention provides a method for constructing a novel mouse model for retinal vein occlusion, which constructs a novel RVO model by combining retinal vein occlusion induced by photodynamic and retinal continuous leakage induced by DL-AAA intravitreal injection; the mouse model for retinal vein occlusion prepared by the construction method is stable, can last for 3-4 days compared with retinal edema constructed by the existing model, can realize more than one month of persistent retinal edema, is convenient to verify a drug target and an action mechanism of a candidate drug after injection, provides a new generation of animal model tool for the research of retinal vein occlusion diseases, is convenient to clarify an RVO pathogenesis and promote the research and development of RVO treatment drugs, and solves the problems in the prior art.
The technical scheme adopted by the invention is as follows:
a method for constructing a novel mouse model with retinal vein occlusion comprises the step of interfering the injection of a Muller cell metabolism inhibitor into the posterior vitreous body of a mouse by adopting a photodynamic method.
Further, the Muller cell metabolism inhibitor is DL-alpha aminocaproic acid solution.
Further, the photodynamic method comprises the steps of injecting the Bengal red solution into the tail vein of the mouse, and immediately irradiating green light with certain wavelength, power and light spot size for a certain time for treatment.
The method for constructing the novel mouse model with retinal vein occlusion specifically comprises the following operation steps:
(1) preparing a bengal solution and a DL-alpha aminocaproic acid solution for later use;
(2) selecting a C57/B6 mouse, anesthetizing, injecting a Bengal red solution into a tail vein, and irradiating two accurately positioned retinal veins at a position away from a visual disc by using argon green laser;
(3) after irradiation, the injection of DL-alpha aminocaproic acid solution in vitreous body is performed immediately to obtain the new mouse model of retinal vein occlusion.
Further, the montage red solution in the step (1) is a montage red solution with the concentration of 8 mg/mL; the DL-alpha aminocaproic acid solution was 0.025M in PBS.
Further, the parameters of the argon green laser irradiation in the step (2) are as follows: wavelength: 532nm, laser spot diameter 50um, laser energy 50mW, duration 3s 2 times or 2s 3 times, interval 0.1s, total 6 s.
Further, the C57/B6 mice in the step (2) are 6-10 weeks old mice; the injection dose of the Bengal red solution is 22-132mg/kg of Bengal red injected into the tail vein of each mouse.
Further, C57/B6 mice were selected as 8-week-old mice; injection dose of the Bengal solution Each mouse was administered with 66mg/kg of Bengal tail vein.
Further, the argon green laser injection in step (2) was performed within 30 seconds after the injection, and the argon green laser was irradiated at a position 500 μm (about 2 disc sizes) from the optic disc.
Further, the dosage of the DL- α -aminocaproic acid solution in the step (3) is 1uL of 0.025MDL- α -aminocaproic acid solution.
The invention has the beneficial effects that:
the preparation method of the mouse model for retinal vein occlusion obtains particularly remarkable experimental effect by performing photodynamic intervention after mouse injection for venous embolism molding and selecting the time points of DL-AAA intravitreal injection and photodynamic molding, and provides an optimal animal model for subsequent research. Specifically, through optimizing an experimental operation flow, the interference of intravitreal injection on eyeball tissues is reduced, the clarity of a refraction medium is kept, and technical parameters such as laser energy, duration and the like in a photodynamic intervention process are adjusted, so that the photodynamic method modeling is smoothly completed, and the construction of a novel animal model is realized; superior animal models were obtained by first analyzing the peak time points of Muller cell loss, retinal leakage and edema following intravitreal injection of DL-AAA, and then selecting the time point at which to combine the photodynamic induction of retinal vein occlusion formation.
Compared with the retinal edema constructed by the existing model which can only last for 3-4 days, the mouse model can realize more than one month of persistent retinal edema, is convenient for verifying drug targets and action mechanisms of candidate drugs after injection, provides a new generation of animal model tools for the research of retinal vein occlusion diseases, is convenient for clarifying RVO pathogenesis and promoting the research and development of RVO treatment drugs, reduces economic burden for patients and improves life quality. The ME secondary to the RVO is the most important field of the biological preparation development in the current ophthalmology field, and the novel RVO model provided by the invention can obviously improve the efficiency of drug development, directly or indirectly promote the industry development and generate economic benefits.
Drawings
FIG. 1 is a process of constructing a novel retinal vein occlusion mouse model of the present invention induced by the photodynamic method of Bengal red light sensitivity combined with DL-AAA intravitreal injection;
FIG. 2 is the construction of a novel RVO mouse animal model of the invention; a is angiography, OCT inspection and retina slice HE staining of the first day after modeling of a normal mouse and an RVO model mouse, and a white arrow indicates a blocked blood vessel; b is retinal thickness analysis before and after model modeling on days 1, 7, 14 and 28 after model RVO mice (n-4, P < 0.001);
FIG. 3 shows the functional verification of Anti-VEGF intervention RVO mouse animal model. Anti-VEGF drug and control PBS intervene in the RVO mouse model and retinal thickness was measured before and after modeling at days 1, 7, 14 and 28, respectively (n-4, P < 0.001).
Detailed Description
In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings.
1. Construction of a novel mouse animal model of RVO:
the method and the material are as follows: constructing a novel RVO model: 8mg/mL Bengal red solution was prepared, 8 weeks old C57/B6 mice were selected, 66mg/kg Bengal red tail vein injection was given to each mouse after anesthesia, and the most accurate two retinal veins were irradiated with an argon green laser at a distance of 500um (about 2 disc sizes) from the optic disc within 30s after injection, with the following parameters: wavelength: 532nm, laser spot diameter 50um, laser energy 50mW, duration 3s 2 times, interval 0.1s, total 6 s. The dry prognosis of the photodynamic method is immediately followed by intravitreal DL-AAA injection. The thicknesses of the retina of the group mouse model were measured after 1, 7, 14 and 28 days after the laser irradiation.
Detection indexes are as follows:
(1) retinal leakage and morphology: form and thickness change of retina are determined through fundus angiography agent OCT;
(2) histopathology: observing changes in retinal-choroidal tissue structure by HE staining, quantifying retinal thickness changes;
(3) and (3) detecting the thickness of the retina: the retinal thickness was analyzed for each set of different time points by OCT detection.
As a result: mouse RVO models prognosis and obvious vascular blockade is found by FFA detection, consistent with the clinical phenotype. Retinal edema was noted following modeling intervention as detected by OCT, and HE staining indicated unclear boundaries between retinal layers of tissue with significant swelling (fig. 2A). By analyzing the retinal thickness before modeling and after 1, 7, 14 and 28 days after modeling (fig. 2B) after the RVO model mouse, the results showed that the retinal thickness significantly increased after 1 day after modeling, and abnormally elevated retinas continued until 28 days after the prediction of stem formation.
And (4) conclusion: the novel RVO mouse model was efficiently established with anatomic and pathological changes consistent with the clinical phenotype, and retinal edema persisted until 28 days post-modeling.
2. Novel RVO animal model effect analysis of anti-VEGF (vascular endothelial growth factor) drug intervention
The method and the material are as follows: the RVO mouse model was constructed as described above, with drug intervention after retinal imaging was completed by P1.
Intervention of anti-VEGF medicine and control group in vitreous cavity injection: commercial anti-VEGF drug Corpescept was purchased and diluted to 2ug/uL for future use. C57/B6 mice of 8 weeks of age were selected, and PBS or 0.5ug/uL or 2ug/uL of combisipu 1uL were injected into the vitreous after anesthesia, respectively. Detection analysis was performed on days 1, 7, 14 and 28 after molding.
As a result: before the intervention of the vitreous drug, the retinal thicknesses of mice in an anti-VEGF intervention group and a control group are not obviously different (n is 4, P is more than 0.05), and the retinal thicknesses of the two groups are obviously increased after the model of the RVO. The intervention with two groups of mice, anti-VEGF and PBS, respectively, was given at P1 and the retinal thickness was measured at P7, P14 and P24, respectively, as shown in FIG. 3, which shows that the anti-VEGF intervention significantly reduced the severity of retinal edema in the RVO model mice.
And (4) conclusion: anti-VEGF effectively inhibits retinal edema in the novel RVO mouse model of the invention.
The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.
The present invention is not described in detail, but is known to those skilled in the art.
Claims (10)
1. A method for constructing a novel mouse model with retinal vein occlusion is characterized by comprising the step of interfering the injection of a Muller cell metabolism inhibitor into the posterior vitreous body of a mouse by adopting a photodynamic method.
2. The method for constructing the novel mouse model of retinal vein occlusion according to claim 1, wherein the Muller cell metabolism inhibitor is DL-alpha aminocaproic acid solution.
3. The method for constructing the novel mouse model for retinal vein occlusion according to claim 1, wherein the photodynamic method comprises the steps of injecting a Bengal red solution into the tail vein of the mouse, and immediately irradiating green light with certain wavelength, power and spot size for a certain time for treatment.
4. The method for constructing the novel mouse model of retinal vein occlusion according to any one of claims 1 to 3, comprising the following steps:
(1) preparing a bengal solution and a DL-alpha aminocaproic acid solution for later use;
(2) selecting a C57/B6 mouse, anesthetizing, injecting a Bengal red solution into a tail vein, and irradiating two accurately positioned retinal veins at a position away from a visual disc by using argon green laser;
(3) after irradiation, the injection of DL-alpha aminocaproic acid solution in vitreous body is performed immediately to obtain the new mouse model of retinal vein occlusion.
5. The method for constructing the novel mouse model for retinal vein occlusion according to claim 4, wherein the Bengal solution of step (1) is a Bengal solution having a concentration of 8 mg/mL; the DL-alpha aminocaproic acid solution was 0.025M in PBS.
6. The method for constructing the novel mouse model for retinal vein occlusion according to claim 4, wherein the parameters of the argon green laser irradiation in the step (2) are as follows: wavelength: 532nm, laser spot diameter 50um, laser energy 50mW, duration 3s 2 times or 2s 3 times, interval 0.1s, total 6 s.
7. The method for constructing a novel mouse model for retinal vein occlusion according to claim 4, wherein the mice of step (2) C57/B6 are 6-10 weeks old; the injection dose of the Bengal red solution is 22-132mg/kg of Bengal red injected into the tail vein of each mouse.
8. The method for constructing a novel mouse model for retinal vein occlusion according to claim 7, wherein the C57/B6 mouse is 8 weeks old; injection dose of the Bengal solution Each mouse was administered with 66mg/kg of Bengal tail vein.
9. The method for constructing a novel mouse model of retinal vein occlusion according to claim 4, wherein the argon green laser injection in step (2) is performed within 30s after the injection, and the argon green laser is irradiated at a position 500 μm away from the optic disc.
10. The method for constructing a novel mouse model of retinal vein occlusion according to claim 1, wherein the dosage of the DL- α -aminocaproic acid solution in the step (3) is 1uL of 0.025M DL- α -aminocaproic acid solution.
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