Construction method of retinal vein occlusion animal model

Abstract

The invention provides a construction method of a retinal vein occlusion animal model, and belongs to the technical field of medicines. The method comprises the following steps of: performing eyeball surface anesthesia on an anesthetized non-human primate animal, puncturing an eyeball after disinfection to establish two sclera tunnels, respectively inserting an illumination optical fiber and an intraocular laser optical fiber into the two sclera tunnels, then injecting a photosensitizer through limb vein, immediately performing laser photocoagulation on the retinal vein under illumination of the illumination optical fiber after injection, and performing laser photocoagulation on the retinal vein under the illumination of the illumination optical fiber. And obtaining the retinal vein occlusion animal model. According to the method, the retinal vein occlusion model can be successfully constructed, secondary macular edema and vascular recanalization are carried out for a long time and can be maintained for 4-5 weeks, the manifestation sign of retinal hemorrhage after retinal vein occlusion is close to that of human beings, and the method has clinical drug responsiveness, shows the clinical research value and has good application prospects. And a good research model is provided for research in the aspects of etiology, pathology and therapeutics.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a method for constructing an animal model of retinal vein occlusion. Background Technology

[0002] Retinal vein occlusion (RVO) is the second leading cause of vision loss due to vascular disease. Based on the location of the retinal vein occlusion, RVO can be classified into central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO). Clinically, retinal vein occlusion often induces complications such as macular edema and neovascularization, leading to visual impairment and blindness.

[0003] Existing animal models of retinopathy of retinal tract (RVO) mostly use rodents (such as rats and mice) or rabbits, constructed through methods such as direct laser photocoagulation, vascular ligation, or drug induction. However, these models have significant limitations: rodents have small eyeballs and lack macular structures, making it difficult to simulate human macular edema; for example, the rabbit retinal vascular system differs greatly from that of humans, and is prone to retinal thermal damage, high vascular recanalization rates, or inconsistencies with the human RVO formation mechanism. Non-human primates (such as cynomolgus monkeys and rhesus monkeys) are considered ideal model animals due to their high similarity to humans in retinal anatomy, blood supply system, and macular characteristics.

[0004] Existing methods for establishing RVO models mainly include vascular ligation, diathermy, intravitreal injection of endothelin-1 (ET-1), laser photocoagulation to block retinal veins, and intravenous injection of photosensitizing drugs combined with laser photocoagulation. Vascular ligation causes obstruction of retinal venous return by burning and ligating the central retinal vein, resulting in prolonged recanalization time. However, the modeling process is complex, technically challenging, highly destructive, and prone to postoperative infection leading to animal death. Diathermy allows for precise location of blood vessels, inducing vasoconstriction and damage through electrothermal stimulation, resulting in immediate vascular occlusion. However, this method uses electrothermal energy to block blood vessels and damages the vascular endothelium, which differs from the human RVO formation mechanism, and its clinical significance requires further research. Endothelin-1 is a potent vasoconstrictive peptide; intravitreal injection of different concentrations of ET-1 elicits different vascular responses. RVO induced by appropriate concentrations of ET-1 is due to severe vasospasm and is only suitable for studying electrophysiological changes in monkeys with retinal ischemia. Laser photocoagulation to block retinal veins... The first method is a non-invasive procedure where a laser is directly applied to the intended blocked blood vessel using a three-mirror lens. The heat energy causes the vessel to become blocked. However, the recanalization rate is high, but the recanalization time is relatively early, requiring repeated photocoagulation. This method can easily cause severe thermal damage to the retinal pigment epithelium and photoreceptor cells, and is often accompanied by vitreous hemorrhage due to vessel wall damage, affecting fundus observation. The second method involves intravenous injection of photosensitizing drugs combined with laser photocoagulation. Photosensitizing drugs are injected into a monkey, and a laser is used to irradiate the retinal vessel to be blocked. Under the combined action of the photosensitizing drug and the irradiated laser, an RVO model is formed. This method creates a model immediately, and the blocked vessel recanalizes in about 7 days. However, the model duration is short, and the direct laser photocoagulation of the fundus vessels through the cornea, anterior chamber, and lens from outside the eye can easily cause excessive damage to the normal retinal tissue surrounding the vessels.

[0005] Therefore, it is essential to establish animal models with good reproducibility that can exhibit pathological processes similar to those of RVO disease for etiological, pathological, and therapeutic research. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing an animal model of retinal vein occlusion.

[0007] This invention provides a method for constructing an animal model of retinal vein occlusion, comprising the following steps: first, anesthetizing the surface of the eyeball of a non-human primate, then disinfecting the eye skin, eyelid margin and conjunctival sac, then puncturing the eyeball to create two scleral tunnels, inserting an illumination fiber and an intraocular laser fiber into the two scleral tunnels respectively, then injecting a photosensitizer via a limb vein, and immediately performing laser photocoagulation on the retinal vein under the illumination of the illumination fiber, thus obtaining the animal model of retinal vein occlusion.

[0008] Furthermore, the photosensitizer is administered in a single injection at a dosage of 15-25 mg / kg.

[0009] Furthermore, the dosage of the photosensitizer is 20 mg / kg.

[0010] Furthermore, the photosensitizer includes Bengal Red, Erythrosine B, or Verteporfin.

[0011] Furthermore, the non-human primates mentioned include cynomolgus monkeys or rhesus monkeys.

[0012] Furthermore, the laser exposure time is 0.1-0.2s, the laser power is 100-200mW, and the laser wavelength is 530-535nm.

[0013] Furthermore, the laser exposure time is 0.15s, the laser power is 160mW, and the laser wavelength is 532nm.

[0014] Furthermore, the laser photocoagulation is performed on the primary and / or secondary branches of the superior retinal vein, and / or on the primary and / or secondary branches of the subretinal vein.

[0015] Furthermore, the laser photocoagulation involves performing laser photocoagulation on the primary and secondary branches of the superior retinal vein and the primary and secondary branches of the subretinal vein. The photocoagulation is performed from the proximal end to the distal end, starting at a distance of 0.5-1.5 optic disc diameters from the optic disc. The length of the primary branch vein receiving photocoagulation is 3.5-4.5 optic disc diameters, and the length of the secondary branch vein is 1.0-2.0 optic disc diameters, avoiding the accompanying artery and the fovea of ​​the macula. The secondary branch vein is a secondary branch vein that faces the macula.

[0016] Furthermore, the laser photocoagulation is performed from the proximal end to the distal end, starting from a distance of one optic disc diameter from the optic disc. The length of the primary branch vein receiving photocoagulation is four optic disc diameters, and the length of the secondary branch vein is 1.5 optic disc diameters, avoiding the accompanying artery and the fovea of ​​the macula; the secondary branch vein is a secondary branch vein that faces the macula.

[0017] The retinal vein occlusion animal model constructed in this invention belongs to the retinal branch vein occlusion model. It employs a method of creating two scleral tunnels through ocular puncture, inserting an illumination fiber and an intraocular laser fiber respectively, and finally injecting a photosensitizer via a limb vein. Immediately after injection, laser photocoagulation is performed on the retinal veins. This method successfully constructs the retinal vein occlusion model, resulting in secondary macular edema and a relatively long vascular recanalization time. Specifically, this method allows for precise targeting of the primary and secondary branches of the superior and inferior retinal veins for laser photocoagulation, minimizing damage to the retina surrounding the photocoagulated vessels. The operation is more precise, and if incomplete venous blood flow occlusion or signs of blood supply recovery are observed during photocoagulation, continuous photocoagulation can be continued at the original laser coagulation site, significantly reducing blood flow recanalization and prolonging the model maintenance time. Furthermore, the retinal hemorrhage manifestations after retinal vein occlusion in this invention are close to those in humans and exhibit clinical drug responsiveness, demonstrating its clinical research value and providing a good research model for etiological, pathological, and therapeutic studies.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 These are color fundus photographs taken two days after the establishment of the retinal vein occlusion model in a rhesus monkey (number 1M002): A is the right eye (OD, model eye), and B is the left eye (OS, normal eye).

[0021] Figure 2 These are color photographs of the fundus of a rhesus monkey (number 1F001, OD) with retinal vein occlusion, where the fundus could not be imaged due to excessive damage during modeling.

[0022] Figure 3 These are color fundus photographs taken 4 weeks after the establishment of the retinal vein occlusion model in rhesus monkeys (number 1M002): A is the right eye (OD) model eye, and B is the left eye (OS) normal eye.

[0023] Figure 4 These are color fundus photographs taken 2 days after the establishment of the retinal vein occlusion model in rhesus monkeys (number 1F002): A is the right eye (OD) model eye, and B is the left eye (OS) model eye + intraocular injection of aflibercept.

[0024] Figure 5 These are color fundus photographs of a rhesus monkey (number 1F002) nine days after retinal vein occlusion modeling: A is the right eye (OD) model eye, and B is the left eye (OS) model eye + aflibercept intraocular injection eye.

[0025] Figure 6 These are color fundus photographs taken 4 weeks after the establishment of the retinal vein occlusion model in rhesus monkeys (number 1F002): A is the right eye (OD) model eye, and B is the left eye (OS) model eye + intraocular injection of aflibercept.

[0026] Figure 7 These are FFA (Fluorescence Analysis) images from a rhesus monkey (number 1M002) retinal vein occlusion model, taken 2 days after modeling: A and B are early and late FFA images of the right eye (OD) model eye, respectively; C and D are early and late FFA images of the left eye (OS) normal eye, respectively.

[0027] Figure 8 These are FFA (Fluorescence Analysis) images of a rhesus monkey (number 1M002) retinal vein occlusion model 4 weeks after modeling: A and B are early and late FFA images of the right eye (OD) model eye, respectively; C and D are early and late FFA images of the left eye (OS) normal eye, respectively.

[0028] Figure 9 These are FFA (Fluorescence Analysis) images from a rhesus monkey (number 1F002) retinal vein occlusion model, taken 2 days after modeling: A and B are early and late FFA images of the right eye (OD), respectively; C and D are early and late FFA images of the left eye (OS) after modeling and intraocular injection of aflibercept, respectively.

[0029] Figure 10 These are FFA (Fluorescence Facial Analysis) images of a rhesus monkey (number 1F002) retinal vein occlusion model 9 days after modeling: A and B are early and late FFA images of the right eye (OD) model eye, respectively; C and D are early and late FFA images of the left eye (OS) model eye after modeling and injection of aflibercept intraocular injection, respectively.

[0030] Figure 11 These are FFA (Fluorescence Facial Analysis) images of a rhesus monkey (number 1F002) retinal vein occlusion model 4 weeks after modeling: A and B are early and late FFA images of the right eye (OD) model eye, respectively; C and D are early and late FFA images of the left eye (OS) model eye after modeling and injection of aflibercept intraocular injection, respectively.

[0031] Figure 12 This is an OCT image taken 2 days after the establishment of a retinal vein occlusion model in a rhesus monkey (number 1M002, OD).

[0032] Figure 13This is an OCT image taken 9 days after the establishment of a retinal vein occlusion model in a rhesus monkey (number 1M002, OD).

[0033] Figure 14 This is an OCT image taken 4 weeks after the establishment of a retinal vein occlusion model in a rhesus monkey (number 1M002, OD).

[0034] Figure 15 These are OCT images taken 2 days after the establishment of the retinal vein occlusion model in a rhesus monkey (number 1F002): A and B are the model eye (OD) of the right eye and the left eye (OS) of the left eye after modeling and intraocular injection of aflibercept, respectively.

[0035] Figure 16 These are OCT images of a rhesus monkey (number 1F002) retinal vein occlusion model 9 days after modeling: A and B are the right eye (OD) model eye and the left eye (OS) model eye after aflibercept intraocular injection, respectively.

[0036] Figure 17 These are OCT images of a rhesus monkey (number 1F002) retinal vein occlusion model 4 weeks after modeling: A and B are the right eye (OD) model eye and the left eye (OS) model eye + aflibercept intraocular injection eye, respectively.

[0037] Figure 18 This is a schematic diagram of photocoagulation points in the construction of the cynomolgus monkey retinal vein occlusion model.

[0038] Figure 19 These are fundus photographs and fluorescein angiography (FFA) images of the cynomolgus monkey retinal vein occlusion model one week after modeling: A and B are fundus photographs of the right eye (OD) model eye and the left eye (OS) normal eye, respectively; C and D are early and late FFA images of the right eye one week after modeling; E and F are early and late FFA images of the left eye one week after modeling.

[0039] Figure 20 A is a fundus photograph taken 2 weeks after the establishment of the cynomolgus monkey retinal vein occlusion model; Figure 20 B is a fundus photograph taken 5 weeks after the establishment of the cynomolgus monkey retinal vein occlusion model; Figure 20 C and 20D are early and late FFA images taken 5 weeks after the establishment of the cynomolgus monkey retinal vein occlusion model, respectively.

[0040] Figure 21 This is an OCT image of a cynomolgus monkey retinal vein occlusion model one week after modeling.

[0041] Figure 22 This is an OCT image of a cynomolgus monkey retinal vein occlusion model two weeks after modeling.

[0042] Figure 23This is an OCT image of a cynomolgus monkey retinal vein occlusion model four weeks after modeling.

[0043] Figure 24 This is an OCT image of a cynomolgus monkey retinal vein occlusion model 5 weeks after modeling.

[0044] Figure 25 This is a graph showing the results of cytokine level detection in the aqueous humor of cynomolgus monkeys before modeling and at 6, 13, 20, and 43 days after modeling.

[0045] Figure 26 This is a histopathological examination image of a cynomolgus monkey retinal vein occlusion model. Detailed Implementation

[0046] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0047] Example 1: Construction of a rhesus monkey retinal vein occlusion model

[0048] Construct it as follows:

[0049] Seven rhesus monkeys were selected and modeled in two batches. The first batch of modeling (1M002, 1F001) used two animals, half male and half female; the second batch of modeling (1M008, 1M009, 1M010, 1F002, 1F003) used five animals, both male and female.

[0050] After anesthetizing the rhesus monkeys, the ocular surface was anesthetized with oxybuprocaine hydrochloride eye drops. Then, povidone-iodine was used to disinfect the ocular skin, eyelid margins, and conjunctival sac. Subsequently, two scleral tunnels were created by puncturing the ocular ball using a scleral tunneling needle (the scleral tunnels were created by puncturing the pars plana of the ciliary body on the nasal and temporal sides, with an angle of 100-130° between the puncture sites). Illumination fiber and intraocular laser fiber were inserted into the two scleral tunnels, respectively. Finally, 20 mg / kg of Bengal red was injected once via the monkey's limb vein. Immediately after the injection, photocoagulation was performed on the primary and / or secondary branches of the retinal veins. Photocoagulation of the veins began at a distance of one optic disc diameter (PD). The length of the primary branch veins receiving photocoagulation was 4 PD, and the length of the secondary branch veins (photocoagulation was only performed on secondary branch veins facing the macula) was 1.5 PD, avoiding the accompanying arteries and the fovea of ​​the macula. The laser wavelength was 532 nm, and no interval was required between the photocoagulation points. If, during photocoagulation, venous blood flow is not completely blocked or signs of restored blood supply are observed, continue photocoagulation at the original laser site for 5-10 consecutive times. During photocoagulation, the laser spot should completely cover the blood vessel to avoid rupturing it and causing bleeding. The total number of photocoagulation sessions should not exceed 200. After laser modeling, apply 1-2 drops of ofloxacin eye ointment to both eyes to keep the cornea moist and prevent infection.

[0051] The first and second batches of rhesus monkeys were modeled according to the above method. After the second batch of animals was modeled, 2 mg of aflibercept ophthalmic injection was immediately administered to each eye via a single intravitreal injection at a dosage volume of 50 μL / eye (Table 1).

[0052] Table 1. Specific modeling sites, laser energy, exposure time, and eye receiving aflibercept ophthalmic injection (right eye OD, left eye OS).

[0053]

[0054] The following is a characterization of the rhesus monkey retinal vein occlusion model constructed by the above method.

[0055] 1.1 Fundus color photography of a rhesus monkey retinal vein occlusion model

[0056] Following the initial modeling of the first batch of rhesus monkeys, starting on the second day, all animals showing retinal edema affecting the macular region, patchy retinal hemorrhages, abnormal retinal changes, and fundus occlusion were observed. Figure 1 In some eyes, due to severe damage during modeling, extensive retinal hemorrhage obstructed fundus imaging. Figure 2 Four weeks after modeling, the fundus occlusion shadow decreased compared to before, but did not completely disappear, and no retinal edema was observed. Figure 3 ).

[0057] After the second batch of rhesus monkeys were modeled, retinal edema affecting the macula was visible in the modeled eyes starting on the second day. Figure 4 (Area marked in yellow box) Patchy retinal hemorrhage; after modeling and intraocular injection of aflibercept, the retinal edema area was limited to the area around the site of vascular laser photocoagulation, without affecting the macula. Figure 4 (B is the area within the red box).

[0058] Nine days after modeling, partial retinal edema was still observed in the modeled eye. Figure 5 (A, indicated by the yellow arrow), but the swelling has lessened compared to before, and no macular edema has been observed; in the eye treated with modeling and aflibercept intraocular injection, no obvious retinal edema has been observed, but some fundus examinations show abnormal retinal changes around the veins. Figure 5 B (indicated by the red arrow) was caused by the modeling operation; no other obvious abnormalities were observed.

[0059] Four weeks after modeling, the retinal veins in the photocoagulation area of ​​the modeled eye showed a white line-like change. Figure 6 A, indicated by the blue arrow); retinal vein filling was good in the area treated with modeling and intraocular injection of aflibercept (or ocular photocoagulation). Figure 6 B, indicated by the yellow arrow).

[0060] 1.2 Fluorescence angiography (FFA) of a rhesus monkey retinal vein occlusion model

[0061] After the first batch of rhesus monkeys were modeled, on the second day, all animals showed visible fluorescent leakage in the blood vessel walls near the modeling site, significant venous dilation and tortuosity, and fundus occlusion. Figure 7 In some eyes, excessive damage during modeling resulted in significant retinal hemorrhage, preventing image formation at the fundus. Four weeks post-modeling, some eyes still showed fluorescein leakage from the venous walls, but the location and area of ​​leakage were reduced, fundus occlusion decreased, and the area of ​​retinal non-perfusion increased. Figure 8 ).

[0062] After the second batch of rhesus monkeys were modeled, on the second day, fluorescence leakage of blood vessel walls near the modeling site was visible in the modeled eyes. Some retinal branch vessels were tortuous and dilated, there were areas of retinal non-perfusion, and fundus images were obscured. Figure 9 ).

[0063] On the 9th day after modeling, retinal vascular wall fluorescence leakage was still visible in the modeled eye, with no retinal perfusion area. The extent and area of ​​leakage had decreased compared to before, and the capillaries at the terminal veins were tortuous and dilated. Figure 10 (As indicated by the yellow arrow A), collateral circulation is formed. Later, the collateral circulation gradually becomes more complete, and no neovascularization is observed. Figure 10 (AB); No retinal vascular wall fluorescence leakage was observed in the eye after modeling and intravitreal injection of aflibercept, no retinal non-perfusion areas were observed, and the veins were patent. Figure 10 CD).

[0064] Four weeks after modeling, the retinal veins in the photocoagulation area of ​​the modeled eye showed a white line-like change. Figure 11 AB (indicated by the blue arrow); retinal vein filling was good in the ocular photocoagulation area after modeling and intraocular injection of aflibercept. Figure 11 CD (indicated by the red arrow).

[0065] 1.3 OCT examination of a rhesus monkey retinal vein occlusion model

[0066] On the second day after the first batch of rhesus monkeys were modeled, macular cystic edema, low-reflectivity dark areas between the retinal neuroepithelial layers, disordered and discontinuous retinal structures at various levels, bulging of the retinal neuroepithelial layer accompanied by underlying medium-to-high reflectivity signal shadows, and strong high-signal shadows in the vitreous body were observed. Figure 12 Some eyes were unable to form an image due to clouding of the refractive media reaching the fundus. On the 9th day after modeling, retinal edema improved, retinal thickness decreased, but the retinal structure remained disordered and discontinuous, and intraretinal edema persisted. Figure 13 Four weeks after modeling, retinal edema subsided, but the RPE layer structure in the macular region remained disordered. Figure 14 ).

[0067] After the second batch of rhesus monkeys were modeled, on the second day, macular cystic edema, low-reflectivity dark areas between the retinal neuroepithelial layers, disordered and discontinuous retinal structures at various levels, bulging of the retinal neuroepithelial layer accompanied by intermediate to high reflectivity signal shadows below it, and strong high signal shadows in the vitreous body were observed. Figure 15 A); In the eye treated with modeling and intraocular aflibercept injection, only perivascular retinal edema was observed with laser photocoagulation, and the edema did not involve the macular area. Figure 15 B).

[0068] On the 9th day after modeling, a low-reflectivity dark area was still seen between the retinal neuroepithelial layers of the modeled eye (suggesting intraretinal edema), with bulging of the retinal neuroepithelial layer accompanied by a medium-to-high reflectivity signal shadow below it, and no macular edema was observed. Figure 16 A); however, the degree of retinal edema and hemorrhage in the eyes treated with modeling and intraocular injection of aflibercept was reduced compared to before, and in some eyes, retinal cystic edema was reduced and retinal height decreased. Figure 16 B).

[0069] Four weeks after modeling, the fundus of the modeled eye showed disordered and discontinuous retinal structure. Figure 17 A); In the eye treated with modeling and aflibercept intraocular injection, no obvious abnormalities were observed in the retina except for the laser photocoagulation point. Figure 17 B).

[0070] The above experimental results demonstrate that the retinal vein occlusion model induced by intravenous injection of Bengal red blood cells and laser photocoagulation in rhesus monkeys was successfully established. Post-modeling, macular cystic edema, retinal hemorrhage, retinal edema, and the formation of collateral circulation in the retinal vessels were observed, indicating successful model establishment. This model could be maintained for 1-2 weeks, showing a tendency for spontaneous healing after 2 weeks, but failing to return to a normal state. Furthermore, immediate intravitreal injection of the positive control, aflibercept ophthalmic injection, resulted in the reduction of cystic edema and retinal thickness one week after modeling, indicating that the positive control had a therapeutic effect on this type of model within one week. This verifies the clinical drug responsiveness of the model and has clinical research value.

[0071] The first batch of rhesus monkeys, 1F001, experienced a large amount of blood entering the vitreous cavity, causing refractive media opacity and preventing fundus imaging. The second batch of rhesus monkeys showed milder symptoms after modeling compared to the first batch. Therefore, reducing laser energy and exposure time can balance modeling damage and model maintenance time.

[0072] In the second batch of rhesus monkey modeling, when the laser energy was reduced and only the primary and / or secondary branches of the superior retinal veins were treated with laser photocoagulation, no cases of excessive damage to the model leading to massive retinal hemorrhage occurred. However, when only the primary or secondary branches of the superior veins were treated with laser photocoagulation (1M008, 1M010), or when the laser power and exposure time were excessively reduced while the primary and secondary branches of the superior veins were treated with laser photocoagulation (1M009, 1M010), neither of these methods could effectively block blood flow and form a model. Ultimately, it was found that using laser photocoagulation of the superior vein's first-order branches with a power of 150mW and an exposure time of 0.15s could induce typical model signs without causing excessive damage (such as 1F002 and 1F003). However, the extent and duration of retinal edema were reduced and shortened compared to the first batch of modeled rhesus monkeys. In the first batch of rhesus monkeys (1F001), the retinal structure of each layer was still disordered and discontinuous on the 9th day after modeling, and retinal edema still existed. In the second batch of rhesus monkeys (1F002), the macular edema had subsided on the 9th day after modeling, and retinal edema was only present in the retina near the modeling area.

[0073] The above experimental results show that in the construction of the rhesus monkey retinal vein occlusion model, reducing laser energy and exposure time while selecting unilateral veins for photocoagulation can balance model damage and model maintenance time, resulting in better 1F002 and 1F003 models. However, they still have some drawbacks, such as faster self-healing and shorter maintenance time (1-2 weeks). Therefore, based on this, we further optimized the model construction parameters to construct the cynomolgus monkey retinal vein occlusion model.

[0074] Example 2: Construction and characterization of a cynomolgus monkey retinal vein occlusion model

[0075] Construct it as follows:

[0076] After anesthetizing the cynomolgus monkeys, topical anesthesia of the eyeball was performed using oxybuprocaine hydrochloride eye drops. Then, povidone-iodine was used to disinfect the ocular skin, eyelid margins, and conjunctival sac. Subsequently, two scleral tunnels were created via scleral tunneling, with an illumination fiber and an intraocular laser fiber inserted into each tunnel, respectively. Then, a single injection of 20 mg / kg of Bengal red was administered via a limb vein. Immediately after injection, laser photocoagulation was performed on the primary and secondary branches of the superior retinal vein and the primary and secondary branches of the subretinal vein. Photocoagulation was performed from one optic disc diameter away from the optic disc, proceeding proximally to distally. The length of the primary branch vein receiving photocoagulation was four optic disc diameters, and the length of the secondary branch veins (photocoagulation only on secondary branch veins facing the macula) was 1.5 PD, avoiding accompanying arteries and the fovea of ​​the macula (specific photocoagulation points are shown in the image). Figure 18As shown in the diagram, the area within the red irregular box represents the photocoagulation area. If, during photocoagulation, venous blood flow is not completely blocked or signs of restored blood supply appear, continue photocoagulation at the original laser site for 5-10 consecutive times. During photocoagulation, the laser spot should completely cover the blood vessel to avoid rupturing it and causing bleeding. The total number of photocoagulation sessions should not exceed 200. Laser parameters: laser power 160mW, exposure time 0.15s, laser wavelength 532nm.

[0077] The following is a characterization of the cynomolgus monkey retinal vein occlusion model constructed by the above method.

[0078] 2.1 Fundus photography and fluorescein angiography (FFA) of the cynomolgus monkey retinal vein occlusion model at 1, 2, and 5 weeks post-modeling.

[0079] One week after modeling, significant bleeding occurred in the blood vessels used for laser photocoagulation. Large areas of flame-shaped hemorrhage appeared in the retinal area responsible for blood drainage. The macular region of the retina showed a yellow-green tinge. FFA (fiber optic angiography) did not reveal fluorescent filling of the laser photocoagulated vessels. Large areas of retinal non-perfusion and fluorescence obscuration were observed. Dilated branch veins and fluorescence leakage were also observed in the macular region. Figure 19 Starting two weeks after modeling, the above-mentioned signs gradually began to recover. Figure 20 A); Five weeks after modeling, the retinal hemorrhage was completely absorbed, but punctate and patchy yellowish-white changes were visible in the macular region of the retina. The laser photocoagulation vessels showed white line-like changes, indicating that blood flow in these vessels had not been restored. Large areas of non-perfusion were still visible in the FFA, and no fluorescence filling was observed in the laser photocoagulation vessels. Figure 20 (BD); This indicates that the cynomolgus monkey retinal vein occlusion model was successfully constructed and is stable in the long term.

[0080] 2.2 Retinal optical coherence tomography (OCT) examination of the cynomolgus monkey retinal vein occlusion model at 1, 2, and 5 weeks post-modeling.

[0081] One week after modeling, detachment of the retinal neuroepithelium and pigment epithelium was observed in the macular region of the retina, accompanied by a low-reflectivity dark area in between, with increased thickness, indicating retinal edema. Figure 21 From week 2-4 after modeling, the thickness of the macular region of the retina gradually decreases, and the edema gradually subsides. Figure 22 , 23 By week 5, the edema had completely subsided, and the retina had fully adhered and repositioned. However, OCT showed thinning and atrophy of the inner retinal layer, accompanied by a mixture of intermediate and high reflectance signals. Punctate or clump-like high reflectance signals were observed in the region between the inner / outer segment junction (IS / OS layer) and the retinal pigment epithelium (RPE) in the photoreceptor cell layer. Figure 24 This indicates that the cynomolgus monkey retinal vein occlusion model has successfully entered the chronic phase, with long-term stable vascular occlusion, resolution of acute edema complications, and irreversible retinal structural damage caused by long-term ischemia due to retinal vein occlusion.

[0082] 2.3 Detection of cytokine levels in the aqueous humor of cynomolgus monkeys before and at 6, 13, 20, and 43 days after modeling.

[0083] like Figure 25 As shown, the concentration of IL-4 did not change significantly before and after modeling. The expression of IL-6, IL-8, and MCP-1 was low before modeling, but their concentrations increased significantly 6 days after modeling, with statistically significant differences compared to the pre-modeling levels (P≤0.05). From the 13th day after modeling, their expression concentrations decreased significantly, reaching levels comparable to the pre-modeling levels. The concentration of VEGF increased significantly 6 days after modeling (P≤0.05), and from the 13th to the 20th day after modeling, its concentration gradually decreased to the pre-modeling levels. This indicates that the cynomolgus monkey retinal vein occlusion model has completed the natural transition from acute inflammatory damage to chronic repair, and the vascular occlusion is stable in the long term.

[0084] 2.4. Histopathological examination of the retinal tissue in a cynomolgus monkey model of retinal vein occlusion.

[0085] Microscopic observation revealed retinal atrophy and retinal degeneration in the cynomolgus monkey retinal vein occlusion model. Figure 26 A) Hypertrophy of the retinal pigment epithelium ( Figure 26 B) Retinal vascular dilation ( Figure 26 C) indicates that the cynomolgus monkey retinal vein occlusion model was successfully constructed.

[0086] The above experimental results demonstrate that intraocular laser photocoagulation of retinal branch veins in cynomolgus monkeys can lead to significant retinal vein occlusion and retinal hemorrhage, followed by macular edema. Macular edema can last for 4-5 weeks, and the expression of related inflammatory factors in the aqueous humor is also significantly increased. However, starting from the 3rd week after modeling, the expression of related cytokines in the aqueous humor is significantly decreased. This proves that the method of the present invention can successfully construct a long-term and stable retinal vein occlusion model in cynomolgus monkeys, followed by macular edema.

[0087] In summary, the method for constructing a retinal vein occlusion model using intraocular laser photocoagulation, as described in this invention, can successfully construct such a model. It utilizes an intraocular laser fiber to accurately target and photocoagulate the first and / or second-order branches of the retinal vein, minimizing damage to the retina surrounding the photocoagulated vessel. This allows for more precise operation, and if blood flow recanalization is detected during photocoagulation, photocoagulation can be repeated immediately, significantly reducing the risk of recanalization and prolonging the model's duration. Experiments have demonstrated that the retinal vein occlusion model constructed using this method has a long recanalization time, exhibits a pathological process similar to that of retinal vein occlusion, and demonstrates clinical drug responsiveness, showcasing its clinical research value and providing a valuable research model for etiological, pathological, and therapeutic studies. Among them, the construction method of the cynomolgus monkey retinal vein occlusion model is better. The manifestations of retinal hemorrhage after retinal vein occlusion are closer to those of humans than those of the rhesus monkey 1F002 model. There was no excessive damage leading to massive retinal hemorrhage. Moreover, the macular edema lasted longer than that of the rhesus monkey 1F002 model. The rhesus monkey retinal vein occlusion model can only be maintained for 1-2 weeks, while the cynomolgus monkey retinal vein occlusion model can be maintained for 4-5 weeks.

Claims

1. A method for constructing an animal model of retinal vein occlusion, characterized in that, Includes the following steps: First, the non-human primates were anesthetized with topical ocular anesthesia. Then, the ocular skin, eyelid margins, and conjunctival sac were disinfected. Subsequently, two scleral tunnels were created by puncturing the eyeball. An illumination fiber and an intraocular laser fiber were inserted into the two scleral tunnels, respectively. Then, a photosensitizer was injected via a limb vein. Immediately after the injection, laser photocoagulation was performed on the retinal veins under the illumination of the illumination fiber, thus obtaining an animal model of retinal vein occlusion.

2. The construction method according to claim 1, characterized in that, The photosensitizer is to be injected once, with a dosage of 15-25 mg / kg.

3. The construction method according to claim 2, characterized in that, The dosage of the photosensitizer is 20 mg / kg.

4. The construction method according to any one of claims 1-3, characterized in that, The photosensitizers mentioned include Bengal Red, Erythrosine B, or Verteporfin.

5. The construction method according to claim 1, characterized in that, The non-human primates mentioned include cynomolgus monkeys or rhesus monkeys.

6. The construction method according to claim 1, characterized in that, The laser exposure time is 0.1-0.2s, the laser power is 100-200mW, and the laser wavelength is 530-535nm.

7. The construction method according to claim 6, characterized in that, The laser exposure time is 0.15s, the laser power is 160mW, and the laser wavelength is 532nm.

8. The construction method according to claim 1, characterized in that, The laser photocoagulation refers to laser photocoagulation of the primary and / or secondary branches of the superior retinal vein, and / or laser photocoagulation of the primary and / or secondary branches of the subretinal vein.

9. The construction method according to claim 8, characterized in that, The laser photocoagulation described herein is performed on the primary and secondary branches of the superior retinal vein and the subretinal vein. Photocoagulation is performed from the proximal end to the distal end, starting at a distance of 0.5-1.5 optic disc diameters from the optic disc. The length of the primary branch vein receiving photocoagulation is 3.5-4.5 optic disc diameters, and the length of the secondary branch vein is 1.0-2.0 optic disc diameters, avoiding the accompanying artery and the fovea of ​​the macula. The secondary branch vein is the secondary branch vein that faces the macula.

10. The construction method according to claim 9, characterized in that, The laser photocoagulation is performed from the proximal end to the distal end, starting from a distance of one optic disc diameter from the optic disc. The length of the primary branch vein receiving photocoagulation is four optic disc diameters, and the length of the secondary branch vein is 1.5 optic disc diameters, avoiding the accompanying artery and the fovea of ​​the macula. The secondary branch vein is a secondary branch vein that faces the macula.

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