KR20200005425A - A novel preparation method of retinal degeneration animal dog model using sodium iodate and retinal degeneration animal dog model using the same - Google Patents

Abstract

The present invention relates to a method for generating a canine animal model with retinal degeneration, comprising the following steps: a) performing pars plana vitrectomy in the eyeball of a dog; and b) injecting sodium iodate (SI) into the eyeball of the dog. The method of the present invention is suitable for generating a hereditary retinal degeneration model.

Description

A novel preparation method of retinal degeneration animal dog model using sodium iodate and retinal degeneration animal dog model using the same}

The present invention relates to a method for producing a retinal degeneration canine animal model using Sodium Iodate (SI) and a retinal degeneration dog animal model using the same.

Retinal degeneration is a disease in which vision is deteriorated due to degeneration of visual cells and eventually blindness, and there is no effective treatment yet. Retinal degeneration in order to apply gene therapy, stem cell therapy, and artificial retina, which are developed for the treatment of diseases such as hereditary retinal degeneration and retinitis pigmentosa (RP) that damage the retina's eye cells and lead to blindness Development of experimental animals with disease is essential.

As such, genetically modified animal models or models for inducing degeneration with drugs have been developed to apply various treatment methods. In animal models of drug-induced retinal degeneration, once drug degeneration protocols are established, they are less expensive than genetically modified animal models, and shorter periods of degeneration can cause degeneration in large quantities and free the investigator from various regulations. There is an advantage.

Sodium Iodate (SI), one of the drugs currently used to make retinal degeneration animals, is a retinal toxicant that primarily damages the retinal pigment epithelium and secondaryly damages other layers of the retina, causing vision loss. . This toxic effect of SI has been used as a model inducing agent for various diseases with pathological and ecological degeneration similar to retinal degeneration. These diseases include age-related Macular Degeneration (AMD), congenital retinal degeneration and Pigmentosa retinitis.

In the past, retinal degeneration was observed by electrophysiological, anatomical and histopathological changes in rabbits treated with 10 mg systemic iodine chloride (NaIO ₃ ) and 20 mg systemic body weight per rabbit. There was also a study showing that degeneration of retinal pigment epithelium and photoreceptor cells and overall retinal degeneration resulted in decreased vision. However, this is a systemic injection of iodine salt, in which case the systemically administered drug affects both eye cells, causing binocular blindness in the test subjects. There is inadequate difficulty in

On the other hand, dogs have similar human and physiological characteristics and similar disease development patterns in humans, in addition to the advantages of being familiar with humans, being easy to handle and training to control and communicate behaviors compared to other animals. Because of this, it is attracting attention as an appropriate animal model. In fact, the number of genetic diseases applicable to human disease research is 224, compared to 65 pigs or 136 cats. In addition, the structure of the eye is anatomically very close to humans compared to other animals, providing many advantages as an animal model.

Against this background, the inventors performed vitrectomy prior to injecting SI into the eye, causing retinal degeneration only in one eye, and inducing an animal model of uniform degeneration throughout the retina, or loss of conical cells of the retina, The present invention has been completed by discovering that only a cell layer comprising both cells and rod cells can produce a selectively lost animal model.

The present invention is to solve the problems of the prior art, the object of the present invention is limited to one eye, to provide a method of manufacturing a retinal degeneration dog animal model induced uniform degeneration throughout the retina and a dog animal model using the same will be.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the invention, a) performing intraocular vitrectomy; And b) injecting Sodium Iodate (SI) into the eye of the dog undergoing vitrectomy; Provided, a method of producing a retinal degeneration dog animal model.

According to one side, the SI may be a concentration of 18mg / ml to 26mg / ml.

According to one side, the SI may be a dilution of 0.9mg to 1.3mg of SI in 0.05ml injection solution.

According to one side, the SI may be injected into the vitreous cavity of the eye.

According to one side, the step b) may be performed after 6 days to 10 days from the step a).

According to one side, the animal model may cause uniform degeneration throughout the retina.

According to one side, the animal model may be degeneration of cone cells of the retina.

According to one side, the animal model may be lost in the cell layer including the cone and rod cells of the retina.

Genetic retinal degeneration model of the retinal degeneration dog model of the present invention by performing a vitrectomy prior to the injection of SI, causing uniform degeneration of the entire retina compared to the case without performing vitrectomy, genetic retinal degeneration model such as retinal pigment degeneration It is suitable for the production of, and can selectively lose the cone cell, or the cell layer containing the cone and rod cells.

In addition, the animal model of the dog produced by the manufacturing method of the present invention can be advantageously used for the development of artificial retina, stem cell treatment research, etc. by inserting artificial retinal device because the structure of the eye is anatomically close to human and the volume is large. Can be.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a wide-field fundus image and autofluorescence image of a rabbit eye according to the presence of vitrectomy and SI concentration.
FIG. 2 is a near infrared (IR) photograph, an optical coherence tomography (OCT) photograph, and H & E tissue staining photograph 1 month after SI injection into a rabbit eye. FIG.
Figure 3 shows the wide-field fundus image, autofluorescence image, near-infrared image, light interference tomography (OCT) and EGR test results of rabbit eye when SI 0.4mg / 0.05ml was injected after vitrectomy.
4 is a wide-field fundus image and autofluorescence image of a rabbit eye intraocularly injected without performing vitrectomy.
FIG. 5 shows optical coherence tomography (OCT), near-infrared (IR) fundus photographs after 2 weeks of intraocular vitreous injection of 0.05 mg (0.8 mg) intraocularly after 2 weeks of vitrectomy with a drug of 16 mg / ml into the dog eye. Retinal potential.
FIG. 6 shows optical coherence tomography (OCT) and near-infrared light at 2 weeks, 4 weeks, and 12 weeks after intravitreal intravitreal injection of 0.05 ml (1.0 mg) of intravenous intravitreal injection of 20 mg / ml of the drug into the dog eye. IR) fundus photograph and retinal potential.
FIG. 7 shows optical coherence tomography (OCT), near-infrared radiation at 2 weeks, 7 weeks, and 12 weeks after intravitreal intravitreal injection of 0.05 ml (12.0 mg) of intravenous intravitreal injection of 24 mg / ml of the drug in the dog eye. IR) fundus photograph and retinal potential.
FIG. 8 shows H & E staining of retinal tissues in dogs comparing dogs with 0.05 mg (12.0 mg) intravitreal intravitreal injection 2 weeks after vitrectomy with 20 mg / ml drug. to be.
9 is a schematic diagram comparing a subretinal injection (A) and an intravitral injection (B) method of SI.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments so that the scope of the patent application is not limited or limited by these embodiments. It is to be understood that all changes, equivalents, and substitutes for the embodiments are included in the scope of rights.

The terminology used herein is for the purpose of description and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and duplicate description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

The present invention finds that when SI is injected without vitrectomy, no degeneration or irregularity occurs in the entire retina, whereas vitrectomy prior to injection of SI can cause uniform degeneration throughout the retina. It is completed.

According to one embodiment of the invention, a) performing intraocular vitrectomy; And b) injecting Sodium Iodate (SI) into the eye of the dog undergoing vitrectomy; Provided, a method of producing a retinal degeneration dog animal model.

"Pars Plana Vitrectomy" (PPV) herein is a surgical procedure consisting of mechanically removing the liquid in the vitreous body by mechanically cutting and aspirating vitreous fluid through a surgical device called a vitrectomy. Vitrectomy can be classified according to the thickness of the vitrectomy needle. Currently, there are 27, 25, 23, and 20 gauge types of vitrectomy needles, and 23G vitrectomy 25 G vitrectomy is most commonly used.

Vitrectomy in the canine animal model production method of the present invention is performed after the scleral incision (trocar insertion), insertion of infusion cannula, control of the perfusion pressure using a balanced salt solution, PVD (Posterior vitreous detachment) Vitreous detachment) induction, retinal torsion treatment steps, and some steps may be omitted as needed.

SI is preferably injected at a concentration of 18 mg / ml to 26 mg / ml, more preferably at a concentration of 20 mg / ml. Injecting SI at less than 18 mg / ml does not cause retinal degeneration, and injecting more than 26 mg / ml is highly toxic and causes problems such as loss of the cell layer and atrophy of the entire retina. It is not preferred because it is difficult to use as an animal model. In addition, since the volume of the eyeball of the dog is constant about 4 ~ 6ml, it is preferable to dilute and inject the SI of 0.9mg to 1.3mg in 0.05ml injection solution.

On the other hand, SI can be injected into the vitreous cavity of the eye. This can solve problems such as binocular blindness, drug toxicity, etc. that occur when systemic injection, such as intraperitoneal administration, and since SI is injected directly into the eye, there is an advantage that can cause retinal degeneration only in one eye. In addition, as can be seen in Figure 9, unlike injection into the subretinal (retretinal), where only localized degeneration is induced, intraretinal injection of the intravitreal uniform retinal degeneration is possible throughout the eye. In the case of injection of SI under the retina, even if the drug is sufficiently injected, if the retina swells above a certain level, the injected drug will leak out of the needle that pierced the retina, and no further injection is possible.

On the other hand, the SI injection of step b) is preferably carried out 6 to 10 days after the vitrectomy of step a), more preferably 7 to 9 days later. If SI is injected 6 days before vitrectomy, the recovery period after vitreous injury may be too short, resulting in an uneven injection of SI. Injecting SI for more than 10 days from performing vitrectomy takes a long time to manufacture an animal model, which is undesirable in terms of efficiency.

According to the method of manufacturing a dog animal model of the present invention, the dog's eye structure is anatomically close to the human, and the volume is easy to insert the artificial retina device, the dog animal model manufactured through the development of the artificial retina stem It may be advantageously used for cell therapy research.

The retinal degeneration dog animal model prepared by the production method of the present invention may cause uniform degeneration of the entire retina, degeneration of the retinal cone cells, or loss of the cell layer including the retinal cone and rod cells. .

In addition, the retinal degeneration animal model of the present invention can be used in experiments when the pattern of retinal degeneration after SI injection is confirmed by an optical fundus camera or an OCT to meet the purpose of the study.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

The following examples were carried out in accordance with the regulations of the ARVO Declaration, which regulates the use of animals for Ophthalmic and Vision Research.

Example  1: Conducting Vitrectomy

Prepare New Zealand white rabbits ranging from 2.5 to 3.5 kg. The average ocular length of rabbits measured using A-scan was 15.75 mm (range: 15.0-16.5 mm).

The rabbits were anesthetized using alfaxalone (5 mg / kg; Alfaxan®, Vetoquinol, West Sussex, UK) and xylazine (4 mg / kg; Rompun®, Bayer Corp., Shawnee Mission, KA, USA). Two drugs were injected intramuscularly 1 cc each. In case of lack of anesthesia, an additional 0.5cc was injected intramuscularly. The pupil of the rabbit was expanded with 0.5% tropicamide and phenylephrine HCl 2.5% eye drops (Mydrin-P®) and treated with proparacine HCl 0.5% (Alcaine®) topical eye drops.

First, insert two trocars about 4 mm from the limbal portion of the rabbit's cornea using a 23-gauge vitrectomy needle in the rabbit's eye, and inject the equilibrium base solution into one of the trocars to maintain the volume while maintaining the volume. Vitrectomy was performed. At this time, the operation was performed by using the illumination of the surgical microscope and checking the intraocular structure using a direct concave lens to secure the surgical field of view.

Example  2: preparation of SI drugs

Dilute sodium iodate (Sigma-Aldrich, St. Louis, MO, USA) with a molecular weight of 191.89 g / mol to an amount of SI of 0.05 mg, 0.1 mg, 0.3 mg, and 0.5 mg per 0.05 ml of phosphate buffered saline (PBS), respectively. To prepare.

Example  3: Evaluation of Retinal Degeneration Pattern According to SI Concentration

Example  3-1

After one week from the vitrectomy of Example 1, the prepared drugs were injected at about 4 mm from the rabbit corneal limbus using a 30 gauge needle. Experimental Example 1 (0.05mg / 0.05ml), Experimental Example 2 (0.1mg / 0.05ml), Experimental Example 3 (0.3mg / 0.05ml), Experimental Example 4 (0.5mg / 0.05ml) according to the concentration of the injected SI In Comparative Example 1, a group injected with SI 0.5mg / 0.05ml without vitrectomy was performed as Comparative Example 2, and a group in which only vitrectomy was performed.

One month after the SI scan, the pattern of retinal degeneration was shown in FIG. 1 by wide-field fundus imaging and autofluorescence fundus imaging. .

In FIG. 1, the left two rows are photographs before the injection and the right two columns are photographs one month after the injection. a ~ d is a group injected with 0.5 mg / 0.05 ml of SI without vitrectomy (Comparative Example 1). A very long paving stone shaped white lesion on the fundus photograph shows a visual streak of the fundus. It was confirmed as the center. In the autofluorescence, very increased autofluorescence lesions in the shape of paving stones were observed.

e ~ h is a group performed only vitrectomy (Comparative Example 2), it can be confirmed that there is no change.

i to l are the group injected with SI 0.05mg / 0.05ml after vitrectomy (Experimental Example 1). No significant change was observed in both fundus and autofluorescence.

m ~ p was a group injected with SI 0.1mg / 0.05ml after vitrectomy (Experimental Example 2), no significant change was observed in both fundus and autofluorescence.

q-t was a group injected with SI 0.3mg / 0.05ml after vitrectomy (Experimental Example 3). No significant change was observed in both fundus and autofluorescence images.

u ~ x is a group injected with SI 0.5mg / 0.05ml after vitrectomy (Experimental Example 4). After surgery, a bright band was observed along the visual streak area in the fundus photograph. Long, increased autofluorescence lesions were observed.

Figure 2 shows a near-infrared (IR) picture, light interference tomography (OCT) picture, and H & E tissue staining picture in order from the left.

a, b is a group injected with 0.5 mg / 0.05 ml of SI without vitrectomy (Comparative Example 1). In the near infrared (IR) image, paving stone-like white lesions were observed on the visual streak area. OCT images of the paving stone showed that the increased reflectors were deposited between the inner and outer layers of the retina, and the surrounding tissues also severely contracted.

c and d were the only vitrectomy group (Comparative Example 2). All of the IR, OCT and histological findings were normal.

e, f is the group injected with SI 0.05mg / 0.05ml after vitrectomy (Experimental Example 1). All of the IR, OCT and tissue findings were normal.

g and h are the group injected with SI 0.1mg / 0.05ml after vitrectomy (Experimental Example 2). All of the IR, OCT and histological findings were normal.

i and j are the group injected with SI 0.3mg / 0.05ml after vitrectomy (Experimental Example 3). All of the IR, OCT and histological findings were normal.

k, l is the group injected with SI 0.5mg / 0.05ml after vitrectomy (Experimental Example 4). In the near-infrared (IR) image, the lesion showed a long elongated reflex in the visual streak area. In the image, the entire retinal layer was observed to be atrophy. The biopsy also revealed severe shrinkage of the entire layer.

m is a diagram of the pattern of living and dead cells according to the concentration of SI. The group injected with 0.05 mg / 0.05 ml showed a similar pattern to the group not injected with SI, indicating that no retinal degeneration was induced. In the group injected with 0.5 mg / 0.05 ml, most of the cells were lost. Can be.

Based on the above results, when too much SI was administered (Comparative Example 1, Experimental Example 4), the entire retinal layer was lost, and when too little SI was administered (Experimental Examples 1 to 3). It can be seen that it is not suitable for.

Example  3-2

Two rabbits were prepared, and after one week after vitrectomy as described in Example 1, an SI of 0.4 mg / 0.05 ml was injected at about 4 mm from the rabbit corneal limbus using a 30 gauge needle. And the result is shown in FIG.

Referring to FIG. 3, the first type (left) is a lesion of a long elongated reflex finding observed in the e fundus photograph and the f visual streak region. On the other hand, the normal retinal structure was observed in the remaining areas. In particular, it was confirmed that the rod reaction, a dark adaptation reaction, was significantly reduced in the h ERG test, and conical cells of the retina were lost through the loss of the light reaction in the positive adaptation reaction. This appears to be due to the loss of cones in the retina because they are fewer than the rods.

In the second type (right), an ERG test showed a lesion with long elongated reflexes in the i fundus and j visual streak areas. While lost, the rest of the site was observed to maintain normal retinal structure. In particular, it was confirmed that the cell outer layer was lost in the entire retina through the loss of both the rod reaction and the light response in the dark adaptation reaction in the h ERG test.

Example  4: Retinal Degeneration Without Vitrectomy

Two rabbits were prepared and injected with a 30-gauge needle at concentrations of 0.3 mg / 0.05 ml and 0.4 mg / 0.05 ml without vitrectomy at about 4 mm from the rabbit corneal limbus. 4 weeks after SI injection, retinal degeneration was confirmed and shown in FIG. 4.

As shown in FIG. 4, retinal degeneration was induced at both the 0.3 mg / 0.05 ml and 0.4 mg / 0.05 ml concentrations, but the retinal degeneration did not occur uniformly throughout the eye and the portion where normal degeneration was not induced remained. I could confirm it.

According to the above experimental results, when the vitrectomy is not performed before the SI injection, irregular retinal degeneration is caused, which leads to a problem that cannot cause uniform retinal degeneration throughout the eye.

In conclusion, the results shown in Example 3-1 and Example 4 suggest that vitrectomy prior to the injection of SI is essential to induce even retinal degeneration in the whole eye.

Example  5: Preparation of Dog Animal Models

5-1. Conduct of Vitrectomy

Prepare mixed race dogs ranging from 25 to 35 kg. The average ocular length of the dog measured using A-scan was 21.47 mm (range: 20.82 ~ 22.07 mm).

The dogs were anesthetized using halothane (5 mg / kg; Alfaxan®, Vetoquinol, West Sussex, UK) and xylazine (4 mg / kg; Rompun®, Bayer Corp., Shawnee Mission, KA, USA). The pupils of the dogs were expanded with 0.5% tropicamide and 2.5% phenylephrine HCl eye drops (Mydrin-P®) and treated with proparacine HCl 0.5% (Alcaine®) topical eye drops.

First, three trocars were inserted into the dog's eye using a 23-gauge vitrectomy needle about 4 mm from the limbus of the dog's cornea. Vitrectomy was performed using a car. At this time, one side of the trocar was inserted into the surgical illumination needle to supply the illumination during surgery, the other side was removed by intraocular vitreous using a vitrectomy needle. Surgery was performed to identify intraocular structures using direct contact lens or indirect lens system (Biom system) to secure the surgical field of vision.

5-2. Preparation of SI Drugs

Sodium iodate (Sigma-Aldrich, St. Louis, MO, USA) of molecular weight 191.89 g / mol is prepared by diluting the amount of SI to 0.8 mg, 1.0 mg and 1.2 mg per 0.05 ml of phosphate buffered saline (PBS), respectively. .

5-3. Comparison of Retinal Degeneration with SI Concentration

After one week from the vitrectomy of Example 5-1, the prepared drugs were injected at about 4 mm from the dog corneal limbus using a 30 gauge needle. Next, the baseline of the SI drug and the injection of SI at concentrations of 0.8 mg / 0.05 ml, 1.0 mg / 0.05 ml, and 1.2 mg / 0.05 ml, respectively, are shown in FIGS. 5 to 7. 5 to 7, the upper photo is a near-infrared fundus photograph and light interference tomography (OCT), and the lower photo shows the retinal potential.

As a result, the injection of SI 0.8mg / 0.05ml after vitrectomy did not show any change in the retina in all tests (see FIG. 5), compared to before SI injection (see FIG. 5), but after injection of SI 1.0mg / 0.05ml 4 After 12 weeks, it was confirmed that the retinal outer layer corresponding to the cell layer in OCT was selectively lost (see FIG. 6). In addition, when the SI was injected at a concentration of 1.0mg / 0.05ml, the light response was completely lost in both rod and cone cells in both the dark reaction and the positive reaction in the ERG test, indicating that the cell outer layer was lost in the entire retina. there was.

In the case of SI 1.2mg / 0.05ml injection, the retinal outer layer corresponding to the cell layer was selectively lost in the OCT after injection, and the retinal inner layer was thinner than the SI 1.0mg / 0.05ml injection. See FIG. 7). Similarly, after the ERG test after injection, the light reaction was completely lost in both rod and cone cells in both the dark adaptation reaction and the positive adaptation reaction.

Meanwhile, as a result of comparing the tissue findings after 3 months of injecting SI at a concentration of 1.0 mg / 0.05 ml with the control group without any treatment, SI was injected after vitrectomy unlike the control group as shown in FIG. 8. In the group, the outer cell layer of the outer retina was lost.

The above results suggest that a desirable dose of retinal degeneration is possible when a specific dose of SI is injected into the vitreous cavity after vitrectomy in the preparation of an animal model of retinal degeneration.

Although the embodiments have been described with reference to the accompanying drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

Claims (8)

a) performing intraocular vitrectomy of the dog; And
b) injecting a sodium ion (SI) into the eye of the dog undergoing vitrectomy; Including, retinal degeneration dog animal model manufacturing method.
The method of claim 1,
Wherein the SI is in a concentration of 18 mg / ml to 26 mg / ml.
The method of claim 2,
Said SI is 0.9 mg to 1.3mg of SI diluted in 0.05ml injection solution, retinal degeneration dog animal model manufacturing method.
The method of claim 1,
And the SI is injected into the vitreous cavity of the eye.
The method of claim 1,
The step b), 6 to 10 days after the step a), the method of producing a retinal degeneration dog animal model.
The method of claim 1,
The animal model is a method of producing a retinal degeneration dog animal model, the uniform degeneration is induced throughout the retina.
The method of claim 1,
The animal model is a method of producing a retinal degeneration dog animal model, the retinal cone cells are lost.
The method of claim 1,
The animal model is a method of producing a retinal degeneration dog animal model is lost in the cell layer comprising the cone and rod cells of the retina.

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