KR20190113151A - Method for visualizing vascular system of animals - Google Patents

Abstract

According to the present invention, a method for visualizing a vascular system of animals comprises the following steps of: injecting a dyeing solution containing a dyeing agent into a blood vessel of an animal; and dyeing the blood vessel and surrounding tissue of the animal by circulating the dyeing solution.

Description

METHOOD FOR VISUALIZING VASCULAR SYSTEM OF ANIMALS

The present invention relates to a method for visualizing the animal's organs, specifically the blood vessels, so that the anatomical structure of the animal's organs can be more clearly distinguished.

Experiments with large mammals are optimal for evaluating the pathological mechanisms or effects of new therapies or drug treatments on human diseases, but their use in experimental studies is limited. Due to their relatively short developmental period and relatively low cost, murine animals are frequently used as in vivo models. It is very important to understand the exact anatomy of animal experiments. Accurate anatomical modeling is required only when accurate anatomical understanding is involved, and it is also very important in determining pathological changes in disease.

However, murine animals have a relatively small body structure, and the head and neck structure is particularly small and complex, which makes it difficult to identify the anatomical structures of blood vessels and nerves, and to use the general anatomical surgical guide. There is no problem.

In this regard, previous studies have struggled with two major problems. First, the incision performed after organ staining causes trauma to the microvascular system, resulting in staining of the entire incision site, which is a more serious problem when major blood vessels need to be removed. Next, tissue fixation using formaldehyde, attempted to solve the above problem, makes the soft anatomy of various organs difficult to operate, makes it difficult to identify arterial, venous and nerves, and tissues of normal anatomical structure Causes artifacts and distortions after fixation.

Accordingly, there has been a continuing need for improved dyeing methods to compensate for the drawbacks associated with making animal models or dissecting animals to understand their structure. In order to solve this problem, the inventors have studied a dyeing method that can more clearly distinguish the anatomical structure of the head and neck area in animal experiments, and completed the present invention.

(1) Japanese Unexamined Patent Application Publication No. 2011-53119

In the present invention, in making an animal model or identifying an anatomical structure of an animal in an animal experiment, the vascular system of an animal can be more clearly distinguished from the anatomical structure of an animal's organ by staining an animal's organ, specifically, a vascular system. Its purpose is to provide a way to visualize. It is also an object of the present invention to provide a method for visualizing the vascular system of an animal, which compensates for the disadvantages associated with conventional animal tissue fixation.

In order to solve the above problems, the present inventors perfused a dyeing agent such as trypan blue reagent to the blood vessel of the animal, and processed the image obtained therefrom, it was difficult to distinguish by the conventional method due to the small size. It was found that the anatomical structure of the vascular system of a murine animal can be clearly distinguished, and completed the present invention.

In one embodiment, a method for visualizing a vascular system of an animal may include: injecting a dyeing solution including a dye into a blood vessel of the animal; And circulating the staining solution to stain blood vessels and surrounding tissues of the animal.

According to one embodiment of the invention, the dyeing agent may comprise one or more selected from the group consisting of trypan blue, alkian blue and toluidine blue.

According to one embodiment of the invention, the dyeing solution may comprise a mixture of the dye and saline.

According to one embodiment of the invention, the dyeing solution may comprise a dyeing agent in an amount of 0.01% by weight to 0.5% by weight.

According to one embodiment of the invention, the dyeing solution is injected through the right atrium of the animal, the dyeing solution circulated through the blood vessels of the animal may be discharged through the right ventricle of the animal.

According to one embodiment of the present invention, the step of circulating the staining solution may be performed until the ear end, nose end, eye or liver of the animal is discolored.

According to one embodiment of the invention, the step of circulating the dyeing solution may be performed for 1 to 10 minutes.

According to one embodiment of the invention, the circulation may be an extracorporeal circulation.

According to one embodiment of the invention, the animal may be a rat.

According to an embodiment of the present invention, the rat can be selected from the group consisting of mouse, rat, rat, mongolian gerbils, hamster and guinea pig.

In another aspect of the present invention, a method of visualizing a vascular system of an animal may include: injecting a dyeing solution including a dye into a blood vessel of the animal; Circulating the staining solution to stain blood vessels and surrounding tissue of the animal until the ear end, nose end, or liver of the animal is discolored; Obtaining an optical image of blood vessels and surrounding tissues of the stained animal; And processing the optical image.

In another aspect of the present invention, a method of visualizing a vascular system of an animal may include: injecting a dyeing solution containing a dye into a blood vessel of the animal; Primary staining of blood vessels and surrounding tissue of the animal by circulating the staining solution until the ear end, nose end or liver of the animal is discolored; And secondary staining of blood vessels and surrounding tissues of the animal with Eosin Y reagent.

In another aspect of the present invention, a method of visualizing a vascular system of an animal may include: injecting a dyeing solution containing a dye into a blood vessel of the animal; Primary staining of blood vessels and surrounding tissue of the animal by circulating the staining solution until the ear end, nose end or liver of the animal is discolored; Secondary staining of blood vessels and surrounding tissues of the animal with Eosin Y reagent; Obtaining an optical image of blood vessels and surrounding tissues of the stained animal; And processing the optical image.

According to the vascular system visualization method of the animal according to the present invention, in making an animal model or grasping the anatomical structure of the animal in the animal experiment, it is possible to more clearly distinguish the anatomical structure of the vascular system of the animal. In particular, it is possible to clearly distinguish the anatomical structure of the vascular system of a rat, especially the head and neck of the head and neck, which has been difficult to distinguish due to its small size.

The present invention can omit the method of fixing the tissue using formaldehyde or the like, so that the properties of the original tissues and organs can be maintained even after staining.

The present invention can help to select an animal model suitable for research for improving understanding and treatment of human diseases.

1 shows a schematic process of a method for visualizing a vascular system of an animal according to the present invention.
2 to 6 show the anatomical structures of mice, rats, sand rats, hamsters and guinea pigs, respectively, in which the vascular system of the animal is stained according to the present invention.
7 to 9 show the results of analyzing the differences in the structure of the facial nerve, salivary glands and carotid arteries of mice, rats, sand rats, hamsters and guinea pigs identified in accordance with the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, embodiment which concerns on this invention is described in detail with reference to an accompanying drawing. The following description is only for easily understanding the embodiments of the present invention, but is not intended to limit the protection scope.

1 shows a schematic process of a method for visualizing a vascular system of an animal according to the present invention.

Referring to FIG. 1, the method for visualizing a vascular system of an animal according to the present invention includes (a) injecting a dyeing solution including a dye into a blood vessel of the animal.

As used herein, the term "animal" is an object to be observed, and any animal having a vascular system may be included without limitation. For the study of human disease, the animal may be a mammal, for example a rat, a rabbit, a pig, a monkey, a dog, a goat, a sheep, a horse and an animal, and a small murine animal for experimental convenience. have. Murine animals can be selected from the group consisting of mice, rats, mongolian gerbils, hamsters, guinea pigs, morphots, chinchillas, remmings and prairie dogs, and mice, rats, sand rats, hamsters commonly used in animal experiments. Or guinea pig, but is not limited thereto.

Animals herein can be prepared in an anesthetic state. Anesthesia may be appropriately performed depending on the condition of the animal, under methods and methods known in the art.

As used herein, “vascular” or “blood system” refers to a passage through which the blood of an animal can be circulated from the heart to each organ and tissue of the animal, and includes aorta, vena cava, pulmonary artery, pulmonary vein and capillary vessel. do. For example, the blood vessels can be the aorta, the vena cava and the capillaries where extracorporeal circulation occurs.

It is difficult to observe the structure of the vascular system of a small-sized musculature with the naked eye.In particular, the structure of the vascular system of the head and neck of a murine animal having a complex structure is very difficult to distinguish the tissues from the surrounding tissues. It is very difficult to manage animals in animal experiments.

As used herein, the term "" staining agent "is a substance that stains the cells constituting the vascular tissue and surrounding tissues, a substance known in the art as a biostaining agent, such as trypan blue, alkyan blue, toluidine blue, It may include pranin, neutral red, nigrosine, fast green and the like, and preferably include one or more selected from the group consisting of trypan blue, alkian blue and toluidine blue, but is not limited thereto. The dyeing agents are charge-bearing molecules, which can migrate into cells by diffusion and stain the cells by reversible electrostatic bonding to the polysaccharides and proteins of the cells.

In the present specification, "dyeing solution" refers to a mixed solution of a dye and a saline solution, but is not limited thereto.

The staining solution does not comprise a fixative solution, such as a formaldehyde solution, and therefore does not cause deformation of the stained tissue. Thus, despite the staining of blood vessels, it is possible to maintain the same or very similar feel as in performing the actual experimental animal production.

The dyeing solution may comprise 0.01% to 0.5% by weight of a dye. If the concentration of the dye in the dyeing solution is less than 0.01% by weight, the color of the stained tissue is light to obtain the desired visual image, if the concentration of the dye is more than 0.5% by weight, the dye penetrates to the surrounding tissue to the tissue and the surrounding tissue The boundaries with the organization may become unclear. The concentration of the dye in the dyeing solution may be adjusted according to the size of the animal body, for example, when the animal is a mouse, the concentration of the dye in the dyeing solution may be 0.01% to 0.1% by weight, such as 0.05% by weight, the animal In the case of rats, sand rats, hamsters and guinea pigs, it may be 0.05% to 0.3% by weight, such as 0.1% by weight.

Referring to Figure 1, the vascular system visualization method of the animal according to the present invention (b) circulating the staining solution until the animal's ear end, nose end or liver discoloration staining the blood vessels and surrounding tissues of the animal Include.

In the present specification, "circulation" may include in vitro circulation and in vivo circulation, but in vitro circulation is preferred for effective staining.

For the extracorporeal circulation of the dyeing solution, the dyeing solution is injected through the animal's right atrium, and the dyeing solution circulating through the animal's vascular system can be discharged through the animal's right ventricle.

Specifically, for the extracorporeal circulation of the staining solution, the abdomen of the animal prepared in an anesthetic state is opened and fixed, and one end of the tube is connected to the right atrium and the right ventricle of the animal, respectively. The other end of each tube is connected to an extracorporeal circulator, so that the dye solution can be circulated into the animal's vascular system with the power of a motor of the extracorporeal circulator. Alternatively, the dye solution may be supplied through a tube connected to the animal's right atrium, and the fluid connected to the animal's right ventricle may be simply discharged from the finished blood vessel of the animal's vascular system. It is also possible to carry out by pulsation.

In the same way, it is also possible to inject the staining solution through the animal's left atrium, and to carry out the dyeing solution circulating through the animal's vasculature so that it can be discharged through the animal's left ventricle.

In this way, the blood present in the blood vessels of the animal can be discharged together. Therefore, at the end of the visualization method of the animal according to the present invention, all the blood of the animal body is kept in a state of being drained, so that it is possible to prevent pigmentation of the surrounding tissue due to bleeding, which occurred when performing dissection by a conventional method. have. Through this, since the vascular structure to be observed can be accurately distinguished from surrounding tissues, it is possible to more easily carry out the study of the running and lesion of blood vessels in the animal model.

Staining the blood vessels and surrounding tissues of the animal may be performed until the ear end, nose end, eye or liver of the animal by staining solution is discolored. The ear end, the nose end, and the eye are both the end of the animal and at the same time, the site of the blood vessels can be easily seen. It can be inferred that the blood vessels of the whole animal are sufficiently stained by confirming the discoloration thereof.

Staining blood vessels and surrounding tissues of the animal by circulating the staining solution can be performed for 1 to 10 minutes, such as for 5 minutes. If the staining step is less than 1 minute, the staining agent is not enough to stain cells, especially living cells, so that it is difficult to distinguish it from surrounding tissues. This can make it difficult to distinguish it from the surrounding tissues.

Referring to FIG. 1, the method for visualizing a vascular system of an animal according to the present invention includes (c) secondary staining of blood vessels and surrounding tissues of an animal with an Eosin Y reagent.

Secondary staining with the Eosin Y reagent herein is performed by an in vitro circulation method, or by cutting the blood vessels and surrounding tissues stained with the staining solution, and the eosin Y after the staining with the staining solution including the staining agent. By dipping in a reagent. By performing additional secondary staining with Eosin Y reagent, it is possible to more clearly identify not only blood vessels but also their surrounding structures.

Referring to FIG. 1, a method for visualizing a vascular system of an animal according to the present invention includes (d) obtaining an optical image of blood vessels and surrounding tissue of a stained animal.

As used herein, the term “optical image” may be a picture or a moving picture taken by a camera used in bright field observation in the art, for example, a CCD camera.

Referring to FIG. 1, the method for visualizing a vascular system of an animal according to the present invention includes (e) processing the optical image.

By “processing an optical image” herein is meant to modify the optical image obtained by the use of an image analysis program commonly used by those skilled in the art according to the protocol to facilitate analysis. The correction may include extracting or deleting some components in the optical image, and changing or inverting the color and contrast. The image analysis program may use, for example, an image J (Image J, https://imagej.nih.gov, USA), but is not limited thereto if it can achieve the same purpose.

Hereinafter, one or more embodiments will be described in more detail.

Example

Example  1. Experimental subject

For carrying out this example, mice (C57BL / 6J, 10 weeks old, 24.7-27.5 g, n = 3, DBL Co., Korea), rats (F344, 11 weeks old, 225.5-255.5 g, n = 3, Japan SLC) , Inc., Japan), sand mouse (mongolian gerbils, Merionesunguiculatus, 10-11 weeks old, 58.7-70.7g, n = 3, Hallym University Experimental Animal Resource Center, Korea), hamster (Syrian, 8 weeks old, 113.1-121.3g) , n = 3, Japan SLC, Inc., Japan) and guinea pigs (Hartley, 8 weeks old, 422.1-426.7 g, n = 3, Japan SLC, Inc., Japan). Used.

Animals were stored in a facility that maintains a standard and constant environment (specifically, temperature: 22 ± 2 ° C., relative humidity: 55 ± 10%, living cycle: 12 hours during the day and 12 hours at night). Normal rodent pellet feed (Cargill Agri Purina, Korea) and water were self-feeding.

Example  2. Staining Tissue

In order to stain the blood vessels of each animal, it was perfused using trypan blue reagent (trypan blue, Cat. N. T6146, Sigma, USA).

Specifically, animals were anesthetized with 3% isoflurane mixed oxygen (RC2 + Anesthesia System, Lab Etc. inc. USA), followed by saline and trypan blue reagents (mouse: 0.05% by weight, and rats, sand rats). , Hamster, guinea pig: 0.1% by weight) was circulated in the body of the animal for 5 minutes through a perfusion pump system (see Fig. 1 (a)).

If blue staining was observed at the ear end, nose end, eye and / or liver of the animal, the perfusion process was terminated (see FIG. 1B). Later, animals were sacrificed at the end of the procedure with minimal pain.

Because no fixed solution (4% paraformaldehyde) was used, the tissue was not preserved and was fresh. As a result, the vasculature could be divided into a dark blue artery system and a light blue venous system.

Example  3. Anatomy and observation of head and neck of animal body

In the animal body dyed in Example 2, after removing the skin from the tip of the nose to the shoulder level, the entire contour of the specimen was examined. The adipose tissue of the lower neck (inferior neck) was removed except for sweat glands under the chin and under the tongue.

At this time, the facial artery (facial artery, FA) and jugular vein (JV) were identified, and the salivary glands and the jugular vein were removed. Exposed sternocleidomastoid muscle was removed, and the subjacent carotid artery of the infra-thyroid muscle was identified. Next to the lower parathyroid muscle, the common carotid artery (CCA) and the vagus nerve (VN) were identified. Finally, the entire thoracic wall was removed to identify the vertebral artery (VA) and the aorta in the subclavian vessels.

Example  4. Identification of cross-sectional images

In order to better understand the anatomical structure for future researchers, cross-sectional shapes of all species were obtained. In addition to vascular staining achieved through perfusion with trypanblue reagent, staining was performed using Eosin Y reagent (cat.N.E4009, Sigma, USA) to better understand the surrounding structure (Fig. 1 (c) ) Reference).

Example  5. Image Shooting

For imaging of anatomical structures in each animal, a standardized approach was used in all species. In order to compare the anatomical structures of the head and neck of five animals, images taken from the same line of sight including the front, side, and inclined surfaces were obtained. In addition, the exact location and anatomical structure of the origin of the jugular vein (JV), total carotid artery (CCA), internal carotid artery (ICA), external carotid artery (ECA) and vertebral artery (VA) were also documented. . To examine the more delicate structures, ophthalmic instruments (8500 Castroviejo Caliper, Fisher, USA) were used for ablation. To obtain the sharpest images, the microscope objective lens and specimens were placed in parallel (to observe in three dimensions and to determine the exact length and area of the structure) .The Canon EOS 1100D and ZEISS OPMI Images were recorded using 1FC (see FIG. 1D). The image was then analyzed using Image J (https://imagej.nih.gov, USA) (see FIG. 1 (e)).

As a result of the analysis, as shown in Table 1, the anatomy of blood vessels of the following sites in the mouse (FIG. 2), the rat (FIG. 3), the sand rat (FIG. 4), the hamster (FIG. 5) and the guinea pig (FIG. 6), respectively. The structure was confirmed.

mouse Rat Sand rat hamster guinea pig A Front view Front view /
Submandibular gland Front view Front view Front view B parotid parotid parotid Side view Perspective view C Side view Heart of parotid gland (absence) Side view after salivary gland removal Hobby artery Hobby artery D Supply artery to the facial nerve Supply artery to the facial nerve Supply artery to the facial nerve Supply artery to the facial nerve Supply artery to the facial nerve E Jugular vein Jugular vein salivary glands Submandibular gland and
Sublingual gland Front view / salivary glands F My neck
musculature Total carotid artery My neck
musculature My neck
musculature Carotid Artery System G Total carotid artery thyroid Total carotid artery Total carotid artery Thyroid gland H Total carotid artery after trachea removal Aorta and
Total carotid artery Artery Artery and
Optic nerve Thyroid gland and total carotid artery bifurcation I Facial vein Facial vein Perspective view Perspective view Extra jugular vein J Carotid artery branch Jugular vein Jugular vein Jugular branch My jugular vein K Deep carotid artery Total carotid artery thyroid Thyroid gland thyroid L Supplying the vascular system to the brain
Before and after External Carotid and Internal Carotid Arteries Early part of vertebral artery Early part of vertebral artery Early part of vertebral artery

Example  6. Statistical Method

For each species, the ratio of organ size to total neck size was calculated using the Kruskal-Wallis Test (see Table 2 below). The Mann-Whitney test was also applied to the post test. Statistical analysis was performed using SPSS version 21 (SPSS IBM, USA). The significance level was set at p <0.05.

Average
(Standard Deviation) mouse Rat Sand rat hamster guinea pig Age (age) 10 11 10-11 8-9 8 Weight (mg) 24.7-27.5 225.5-255.5 58.7-70.7 113.1 ~ 121.3 422.1-426.7 Marginal mandible
Branch width (cm) 0.047 ± 0.047 0.033 ± 0.033 0.043 ± 0.043 0.043 ± 0.043 0.041 ± 0.0410 Branch width (cm) 0.025 ± 0.025 0.036 ± 0.036 0.041 ± 0.041 0.045 ± 0.045 0.148 ± 0.148 Ball / limit
Mandible 0.556 ± 0.556 1.138 ± 0.138 0.964 ± 0.964 1.064 ± 0.064 3.373 ± 0.373 (Lower Mandible and Tongue Area) / Internal Neck (%) 53.53 ± 8.66 38.53 ± 5.56 34.33 ± 3.20 64.90 ± 13.16 21.87 ± 2.06 Parotid gland area / side neck (%) 23.77 ± 2.72 26.5 ± 6.76 23.63 ± 3.41 13.18 ± 2.99 6.97 ± 3.07 Total carotid artery
(Right prayer angle) 54.23 ± 32.05 70.65 ± 15.05 40.31 ± 13.80 88.77 ± 17.77 67.38 ± 21.32 Total carotid artery
(Left origin angle) 110.63 ± 4.78 129.57 ± 23.14 86.66 ± 22.82 117.89 ± 1.57 141.20 ± 19.23 Carotid angle (inside and outside) 39.96 ± 8.71 28.99 ± 6.27 53.99 ± 4.82 34.22 ± 3.51 28.30 ± 2.96

6.1. Relative Size Comparison of Cervical Structures

To compare the relative size of tissues in five animals, the average value of cervical structures (facial nerves, blood vessels, glands) was calculated using ALTools (http://www.altools.co.kr) (Table above) 2).

6.2. Organ relative size comparison

The widths of the buccal and marginal mandibular branches were calculated, and the width ratio (ball / limit lower) was calculated and compared with each other.

It was confirmed that each species has a specific pattern of neural branches (see A to E of FIG. 7). In mice, the marginal mandibular branches were thicker and the cheeks were thinner than other species (see FIG. 7B).

Mean values of nerve widths of five animals were compared (see F in FIG. 7). The bran branches of guinea pigs were relatively thick compared to other species (p = 0.021). In contrast, there was no significant difference in marginal mandibular branches between species (p = 0.641).

In addition, the ratio of ball width / marginal mandibular width was calculated and compared (see G in FIG. 7). The maximum difference to the mean was seen in the marginal mandibular branch of the mouse and the cheek branch of the guinea pig (p = 0.001). Mice did not have cervical branches of the facial nerve (see H in FIG. 7).

These differences were thought to depend on the dietary habits of each species and the relative degree of bruxism explained by each species.

6.3. Comparison of the structure of salivary glands

Instead of the traditional assessment method (tracheal-to-weight ratio), the ratio of salivary gland size to cervical area was compared.

In order to improve the accuracy, a negative image of the tissue was first produced (see A to J of FIG. 8, yellow line is area calculation line). In the analysis of the relative ratios of area, the parotid glands of hamsters and guinea pigs were relatively small compared to other species (guinea pigs were the smallest) (p = 0.008) (see K in FIG. 8). In contrast, the lower mandible and sublingual glands of hamsters were significantly larger than other species (p = 0.012) (see L in FIG. 8). The hamster had the largest ball pocket.

In this analysis, the area of the oral mucosa was correlated with the area of the lower mandible and sublingual glands, but not the parotid gland. Since most saliva is produced by the parotid glands, it was thought that there was no direct correlation between saliva production and oral mucosa size.

6.4. Comparison of Carotid Artery Structures

To determine the best animal model for the study of atherosclerosis and ischemia, the angles of the different vessels were measured from the origin of the vessels.

To this end, in the present embodiment, the cervical blood vessel image was extracted from the optical image, the branch angle was calculated, and compared with each other (see FIGS. 9A through 9E). The angle between the feed and branch arteries for each species of animal was measured (see F in FIG. 9).

The origin of the total carotid artery from the aorta was not significantly different as a result of the large standard deviation (p = 0.192) (see G in FIG. 9), but the hamster was nearly perpendicular to each side of the neck (see G in FIG. 9). .

Based on the anatomical findings, the hamster was considered to be optimal as an animal model for the study of unilateral atherosclerosis or ischemia, especially when the contralateral control is needed.

In five animals, association with common carotid artery branching was confirmed (see H to L in FIG. 9). Of all species, sand rats had the widest angle (p = 0.033) (see M in FIG. 9).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown by the following claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. Should be.

Claims (13)

Injecting a dyeing solution comprising a dye into a blood vessel of the animal; And
Circulating the staining solution to stain blood vessels and surrounding tissue of the animal
Vascular system visualization method of an animal comprising a. The method of claim 1,
Said staining agent comprises at least one selected from the group consisting of trypan blue, alkian blue and toluidine blue. The method of claim 1,
The dyeing solution is a blood vessel visualization method of the animal, characterized in that it comprises a mixture of the dye and saline. The method of claim 1,
The staining solution comprises a staining agent 0.01% to 0.5% by weight of the vascular system visualization method of the animal, characterized in that. The method of claim 1,
The dyeing solution is injected through the right atrium of the animal, the dyeing solution circulated through the blood vessels of the animal is vascular system visualization method of the animal, characterized in that discharged through the right ventricle of the animal. The method of claim 1,
Circulating the staining solution is performed until the animal's ear end, nose end, eyeball or liver discolor. The method of claim 1,
Circulating the staining solution is performed for 1 to 10 minutes. The method of claim 1,
Said circulation is an extracorporeal circulation. The method of claim 1,
The animal is a vascular system visualization method, characterized in that the animal is a murine. The method of claim 7, wherein
Said murine animal is selected from the group consisting of mouse, rat, sand rat, hamster and guinea pig. Injecting a dyeing solution comprising a dye into a blood vessel of the animal;
Circulating the staining solution to stain blood vessels and surrounding tissue of the animal until the ear end, nose end, or liver of the animal is discolored;
Obtaining an optical image of blood vessels and surrounding tissues of the stained animal; And
Processing the optical image
Vascular system visualization method of an animal comprising a. Injecting a dyeing solution comprising a dye into a blood vessel of the animal;
Primary staining of blood vessels and surrounding tissue of the animal by circulating the staining solution until the ear end, nose end or liver of the animal is discolored; And
Secondary staining of blood vessels and surrounding tissues of the animal with Eosin Y reagent
Vascular system visualization method of an animal comprising a. Injecting a dyeing solution comprising a dye into a blood vessel of the animal;
Primary staining of blood vessels and surrounding tissue of the animal by circulating the staining solution until the ear end, nose end or liver of the animal is discolored;
Secondary staining of blood vessels and surrounding tissues of the animal with Eosin Y reagent;
Obtaining an optical image of blood vessels and surrounding tissues of the stained animal; And
Processing the optical image
Vascular system visualization method of an animal comprising a.

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