KR102224060B1 - A Method for visualizing vascular system of central nerve system - Google Patents

Abstract

The present application relates to a method for staining blood vessels in the central nervous system of an animal, a method for visualizing blood vessels in the central nervous system in an animal, a method of providing information for diagnosing cerebrovascular disease, and a screening method for a therapeutic agent for cerebrovascular disease.
Using the central nervous system blood vessel visualization method of the present application, it is possible to grasp the structure of cerebrovascular and spinal blood vessels, and it is easy to identify variations in blood vessels related to brain diseases such as Willis-hwan artery and basal artery. Alternatively, it may be used for research on therapeutic agents for cerebrovascular diseases.

Description

{A Method for visualizing vascular system of central nerve system}

The present application relates to a method for staining blood vessels in the central nervous system of an animal, a method for visualizing blood vessels in the central nervous system in an animal, a method of providing information for diagnosing cerebrovascular disease, and a screening method for a therapeutic agent for cerebrovascular disease.

In order to evaluate the pathological mechanisms or effects of new therapies or drug treatments for human diseases, experiments with large mammals are optimal, but their use in experimental studies is limited. Due to their relatively short development period and relatively low cost, murine animals are often used as in vivo models. It is very important to understand the exact anatomical structure in animal experiments. Accurate laboratory animal modeling is possible only when an accurate anatomical understanding is accompanied, and it is also very important in determining pathological changes for diseases.

However, the murine animal has a relatively small body structure, and the head and neck structure has a particularly small and complex structure, so it is difficult to identify the anatomical structure of blood vessels and nerves, and a surgical encyclopedia of general anatomy is used. There is a problem that cannot be done.

Previously, it was attempted to observe blood vessels and nerves, especially the vascular structure of the central nervous system, using magnetic resonance salt images, microcomputer tomography, photoacoustic microscopy, continuous two-photon tomography, and multiphoton microscopy, but actual results were used in experimental animals. It is difficult to obtain from.

Accordingly, the present inventors have endeavored to develop a method for visualizing the structures of central nervous system blood vessels, specifically cerebrovascular and spinal blood vessels, and as a result of developing methods and visualization methods for staining blood vessels of the central nervous system of animals, and diagnosing cerebrovascular diseases using this The present invention was completed by developing an information providing method and a screening method for a therapeutic agent for cerebrovascular disease.

Korean Patent Registration No. 10-1124641

The first aspect of the present application, (a) injecting a dye solution containing a dye into the blood vessel of the subject; And (b) circulating the staining solution to stain the blood vessels and surrounding tissues of the individual.

A second aspect of the present application, (a) injecting a dye solution containing a dye into the blood vessel of the subject; (b) circulating the staining solution to stain blood vessels and surrounding tissues of the individual; (c) obtaining an image of the stained brain and spinal cord; And (e) to provide a method for visualizing the blood vessels of the central nervous system of the animal comprising the step of processing the obtained image.

A third aspect of the present application includes the steps of: (a) visualizing the blood vessels of the central nervous system of the individual using the method according to claim 6; And (b) to provide a method for providing information for diagnosing cerebrovascular disease comprising the step of confirming the mutation of the visualized central nervous system blood vessels.

A fourth aspect of the present application includes the steps of: (a) administering a candidate material for cerebrovascular disease treatment to an animal model of cerebrovascular disease; And (b) visualizing the blood vessels of the central nervous system of the animal model of cerebrovascular disease to which the candidate substance was administered by using the method according to claim 6; And (c) to provide a screening method for a therapeutic agent for cerebrovascular disease comprising the step of confirming the degree of variability in the blood vessels of the central nervous system according to the administration of the candidate substance.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be considered that the scope of the present application is limited by the specific description described below.

The first aspect of the present application, (a) injecting a dye solution containing a dye into the blood vessel of the subject; And (b) circulating the staining solution to stain the blood vessels and surrounding tissues of the individual.

According to one embodiment of the present application, the step (a) includes: i) removing blood from the subject; And ii) circulating the staining solution throughout the circulatory system of the individual using a perfusion pump.

The term “animal” as used throughout the specification of the present application is an object to be observed, and may be included without limitation as long as it is an animal having a vascular system. For the study of human diseases, the animals may be mammals, for example, rats, rabbits, pigs, monkeys, dogs, goats, sheep, horses, and may be small-sized murine animals for experimental convenience. have. The murine animal may be selected from the group consisting of mice, rats, gerbils, hamsters, guinea pigs, mormots, chinchillas, lemmings and prairie dogs, and are wild mice, mice, rats, gerbils, hamsters or guinea pigs generally used in animal experiments. It is preferable, but is not limited thereto.

According to one embodiment of the present application, the individual may generally mean all animals that can be used as experimental animals including rabbits, monkeys, pigs, horses, sheep, goats and chickens, and specifically excludes humans. It can be.

According to the exemplary embodiment of the present disclosure, the individual may include a person, but is not limited thereto.

According to the exemplary embodiment of the present disclosure, the individual may be a rodent, and specifically, may be selected from the group consisting of wild Korean mice, mice, rats, gerbils, guinea pigs, and hamsters, but is not limited thereto.

According to one embodiment of the present application, the individual may be prepared in an anesthetized state. Anesthesia can be appropriately performed according to the condition of the animal under methods and usages known in the art.

As used throughout the present specification, the term “blood vessel” or “vascular system” refers to a passage through which blood of an animal can circulate between the organs and tissues of the animal body from the heart, and refers to aorta, vena cava, pulmonary artery, pulmonary veins, and capillaries. For example, blood vessels can be arteries, veins and capillaries associated with the central nervous system.

According to one embodiment of the present application, the central nervous system blood vessels are blood vessels present in the central nervous system, and may be blood vessels that supply blood to the central nervous system, and are specifically present in the brain, cranial nerves, spinal cord, olfactory nerves, brain parenchyma and pituitary gland. Or it may be a blood vessel supplying blood thereto, but is not limited thereto.

According to the exemplary embodiment of the present disclosure, the central nervous system blood vessel may include a Circle of Wills (CoW) artery and a basilar artery (BA).

The term "Circle of Wills, CoW" as used throughout the specification of the present application is a ring-shaped vascular structure that connects the left and right internal carotid arteries in the front and the vertebral basal arteries in the rear, and forms the lateral circulation of the brain. This is the most basic structure. In the case of cerebral aneurysm, degenerative damage of blood vessels due to hemodynamic stress is known as one of the main triggers. Hemodynamics in asymmetric Willis ring such as hypoplasia of the Al segment, fetal circulation of the posterior transit artery, and hypoplasia of the Pl segment. It is known that a cerebral aneurysm is caused by internal stress. In addition, in patients with cerebral aneurysms as well as stenosis or obstruction of cerebral blood vessels, it is known that the development of collateral circulation due to structural mutation of Willis ring can act as a factor causing symptoms. Through this, you can diagnose cerebrovascular-related diseases.

The term "basilar artery (BA)" as used throughout the specification of the present application is a blood vessel that sends oxygen-rich blood from the heart to the brain, and is located at the bottom of the brain where two vertebral arteries merge, and the vertebral artery and Together, they constitute the ulnar basal artery system, and the ulnar basal artery system becomes a part of the Willis ring that supplies blood to the brain. The cerebral basal artery is again divided into left and right posterior cerebral arteries, and serves to supply blood to the brain stem, cerebellum, and occipital lobes. If blood is not sufficiently supplied to the cerebral basal artery, symptoms such as dizziness, dizziness, confusion, and visual impairment may occur, and in severe cases, brain injury or death may result. In the case of the cerebral basal artery, an aneurysm in which the blood vessel is blocked or the blood vessel bulges may occur, and the blood vessel rupture may occur due to the above abnormalities. Related diseases can be diagnosed. In addition, the basal cerebral artery may be used in combination with the basal cerebral artery.

According to one embodiment of the present application, the step of staining the blood vessels and surrounding tissues of the animal by circulating the staining solution may be performed for 1 minute to 10 minutes, specifically for 5 minutes. If the staining step is less than 1 minute, the staining agent is not sufficient to stain cells, especially living cells, making it difficult to distinguish it from surrounding tissues. If it exceeds 10 minutes, the staining target is stained not only blood vessels but also surrounding tissues. It can make it difficult to distinguish between a person and the surrounding tissues.

As used throughout the present specification, the term "dye" refers to a substance that stains vascular tissues and cells constituting the surrounding tissues, and substances known as bio-staining agents in the art, for example, trypan blue, alcian blue, toluidine Blue, safranin, neutral red, nigrosine, fast green, and the like may be included, and it is preferable to include at least one selected from the group consisting of trypan blue and alcian blue, but is not limited thereto. The staining agents are charged molecules, and they migrate into the cell by diffusion and reversibly electrostatically bind to polysaccharides and proteins of the cell, thereby staining cells.

According to the exemplary embodiment of the present application, the dyeing agent may include Alcian Blue or Trypan Blue.

The term "Alcian Blue" as used throughout the specification of the present application is a reagent for dyeing polysaccharides such as hyaluronic acid, and has a cation and binds to the anionic site of the saccharide to undergo a reversible electrostatic bond. However, the electrostatic bond between Alcian Blue and the anion site of the saccharide can be easily broken, and the degree of dyeing varies depending on the degree of this bond, and various types of polysaccharides can be expressed. Polysaccharides are dyed blue or bluish green by Alcian Blue.

According to the exemplary embodiment of the present disclosure, the dyeing solution may include a mixture of a dyeing agent and a saline solution.

According to the exemplary embodiment of the present disclosure, the dyeing solution may include a mixture of a dyeing agent and paraformaldehyde.

According to the exemplary embodiment of the present disclosure, the dyeing solution may include 0.01% to 0.5% by weight of a dye, but is not limited thereto.

A second aspect of the present application, (a) injecting a dye solution containing a dye into the blood vessel of the subject; (b) circulating the staining solution to stain blood vessels and surrounding tissues of the individual; (c) obtaining an image of the stained brain and spinal cord; And (d) it provides a method for visualizing the blood vessels of the central nervous system of the animal comprising the step of processing the obtained image. The content overlapping with the first aspect also applies to the visualization method of the central nervous system blood vessels of the animal on the second aspect.

According to an embodiment of the present disclosure, obtaining an image may be obtaining an image including a stained central nervous system blood vessel using a microscope or various observation equipment, and specifically, a stained central nervous system blood vessel image using a stereoscopic microscope It may be to obtain.

As used throughout this specification, the term "processing an image" means modifying an image obtained to facilitate analysis by using an image analysis program generally used by a person skilled in the art according to a protocol. The modification may include extracting or deleting some components in the image, and changing or inverting color and contrast. The image analysis program may use, for example, Image J (https://imagej.nih.gov, USA), but is not limited thereto as long as it can achieve the same purpose.

According to the exemplary embodiment of the present disclosure, processing the image may include at least one selected from the group consisting of color inversion imaging and cartoon imaging.

According to one embodiment of the present application, the step (a) includes: i) removing blood from the subject; And ii) circulating the staining solution throughout the circulatory system of the individual using a perfusion pump.

According to the exemplary embodiment of the present application, the dyeing agent may include Alcian Blue or Trypan Blue.

According to the exemplary embodiment of the present disclosure, the dyeing solution may include a mixture of a dyeing agent and paraformaldehyde.

According to the exemplary embodiment of the present disclosure, the dyeing solution may include 0.01% to 0.5% by weight of a dye, but is not limited thereto.

According to the exemplary embodiment of the present disclosure, the central nervous system blood vessel may include a Circle of Wills (CoW) artery and a basilar artery (BA).

According to one embodiment of the present application, the individual may generally mean all animals that can be used as experimental animals including rabbits, monkeys, pigs, horses, sheep, goats and chickens, and specifically excludes humans. It can be.

According to the exemplary embodiment of the present disclosure, the individual may include a person, but is not limited thereto.

According to the exemplary embodiment of the present disclosure, the individual may be a rodent, and specifically, may be selected from the group consisting of wild Korean mice, mice, rats, gerbils, guinea pigs, and hamsters, but is not limited thereto.

A third aspect of the present application includes the steps of: (a) visualizing the central nervous system blood vessels of the individual using the method of visualizing the central nervous system blood vessels of the animal; And (b) it provides a method for providing information for cerebrovascular disease diagnosis comprising the step of confirming the mutation of the visualized central nervous system blood vessels. The content overlapping with the first and second aspects also applies to the third aspect of the method of providing information for cerebrovascular disease diagnosis.

According to one embodiment of the present application, the step of confirming the mutation in step (b) comprises comparing the central nervous system blood vessel visualization data of a control group in which no central nervous system vascular mutation or cerebrovascular disease has occurred. Can be.

According to one embodiment of the present application, the cerebrovascular disease may be selected from the group consisting of cerebral aneurysm, cerebral infarction, cerebral thrombosis, cerebral embolism, cerebral hemorrhage and stroke, and specifically cerebrovascular mutation (blockage, narrowing, bursting, bleeding, etc.) , Willis-hwan may be a disease caused by a structural mutation such as segmental amorphism of the artery, a change in thickness, or a blockage of the basal artery, but is not limited thereto.

According to one embodiment of the present application, the mutation of the blood vessels of the central nervous system is various vascular abnormalities such as narrowing or clogging of blood vessels, a case in which blood does not flow smoothly, damage or swelling of blood vessels or blood vessel walls, and bursting of blood vessels. As a sign, it may mean that a cerebrovascular blood vessel is narrowed, blocked, damaged, burst, or bleeding occurs, but is not limited thereto.

As used throughout the present specification, the term "individual" refers to all animals such as rodents, mice, rats, hamsters, pigs, cattle, livestock, including humans suffering from or at risk of cerebrovascular disease herein.

According to the exemplary embodiment of the present disclosure, the individual may be a human, but is not limited thereto.

According to one embodiment of the present application, the individual may be selected from the group consisting of wild Korean mice, mice, rats, gerbils, guinea pigs, and hamsters, but is not limited thereto.

A fourth aspect of the present application includes the steps of: (a) administering a candidate material for cerebrovascular disease treatment to an animal model of cerebrovascular disease; And (b) visualizing the blood vessels of the central nervous system of the subject to which the candidate substance was administered using the method according to claim 6; And (c) it provides a screening method for a therapeutic agent for cerebrovascular disease comprising the step of confirming the degree of variance in the blood vessels of the central nervous system according to the administration of the candidate substance. The content overlapping with the first to third aspects also applies to the fourth aspect of the screening method for a therapeutic agent for cerebrovascular disease.

According to the exemplary embodiment of the present disclosure, the method may further include the step of (d) comparing the blood vessel visualization data of the individual with the central nervous system before administration of the candidate substance.

According to one embodiment of the present application, the method comprises the steps of determining the candidate material as a treatment for cerebrovascular disease when the degree of variability in the blood vessels of the central nervous system is alleviated or symptoms of cerebrovascular disease are alleviated according to the candidate material for cerebrovascular disease treatment. It may be to further include.

According to one embodiment of the present application, the mutation of the blood vessels of the central nervous system refers to various vascular abnormalities such as narrowing or clogging of blood vessels, damage or swelling of blood vessels or blood vessel walls, and bursting of blood vessels. As such, it may mean that bleeding occurs due to narrowing, clogging, damage, or bursting of cerebrovascular blood vessels, but is not limited thereto.

According to one embodiment of the present application, in the case where the degree of variability in the central nervous system blood vessels is alleviated in the method, the structure of the blood vessel is normally restored as compared with the central nervous system blood vessel visualization data of the individual before administration of the candidate substance, or cerebrovascular It may mean that the blocked part of the cerebral blood vessel is punctured, blood flow of cerebrovascular blood vessels is restored, etc., which is similar to the central nervous system vascular structure and blood flow of the control group that does not suffer from cerebrovascular disease.

The step of confirming the degree of symptoms or mutations described above refers to a step of confirming the progress or recovery of the condition. For the purposes of the present application, the step of confirming the degree of symptoms or mutations is a candidate substance capable of treating cerebrovascular disease or mutation of central nervous system blood vessels occurring in an individual in an animal model in which cerebrovascular disease or mutation of central nervous system blood vessels has occurred. After administration, it may be understood as a series of processes of assessing whether the candidate substance can treat the cerebrovascular disease by checking the progress of cerebrovascular disease or central nervous system vascular mutation, but is not particularly limited thereto. . Specifically, when the symptom of the above-described cerebrovascular disease or the degree of variability in the blood vessels of the central nervous system is checked, and the symptom or variability is alleviated, the candidate substance may be determined as a treatment for cerebrovascular disease.

According to one embodiment of the present application, the cerebrovascular disease may be selected from the group consisting of cerebral aneurysm, cerebral infarction, cerebral thrombosis, cerebral embolism, cerebral hemorrhage and stroke, and specifically cerebrovascular mutation (blockage, narrowing, bursting, bleeding, etc.) , Willis-hwan may be a disease caused by a structural mutation such as segmental amorphism of the artery, a change in thickness, or a blockage of the basal artery, but is not limited thereto.

The term "cerebrovascular disease animal model" as used throughout the present specification refers to rodents, mice, rats, hamsters, pigs, cows, livestock, etc., including humans suffering from cerebrovascular diseases or mutations in the blood vessels of the central nervous system. Means all animals.

According to the exemplary embodiment of the present disclosure, the cerebrovascular disease animal model may be selected from the group consisting of wild Korean mice, mice, rats, gerbils, guinea pigs, and hamsters, but is not limited thereto.

According to one embodiment of the present application, the central nervous system blood vessels are blood vessels present in the central nervous system, and may be blood vessels that supply blood to the central nervous system, and are specifically present in the brain, cranial nerves, spinal cord, olfactory nerves, brain parenchyma and pituitary gland. Or it may be a blood vessel supplying blood thereto, but is not limited thereto.

According to the exemplary embodiment of the present disclosure, the central nervous system blood vessel may include a Circle of Wills (CoW) artery and a basilar artery (BA).

Using the central nervous system blood vessel visualization method of the present application, it is possible to grasp the structure of cerebrovascular and spinal blood vessels, and it is easy to identify variations in blood vessels related to brain diseases such as Willis-hwan artery and basal artery. Alternatively, it may be used for research on therapeutic agents for cerebrovascular diseases.

1 is a diagram schematically illustrating a process of staining and processing an image of blood vessels of the central nervous system.
2 is a diagram showing the blood vessel system (vascular distribution) of the brain.
[CoW: Willis-hwan, Basilar A: basal artery, Ant cereb A: anterior cerebral artery, HPS: pituitary-portal system, skull base: cranial base, Anterior direction on anterior-posterior axis: anterior-posterior axis, superior direction on superior-inferior axis: upper direction of upper-lower axis, right direction on right-left axis: right direction of left-right axis]
Figure 3 is a view showing the blood vessel anatomy of the yeonsu, cerebrum and cerebellum.
[Pons: pons, Basilar A: basal artery, cortex: cortex, MCA: middle cerevral arteries, Dorsum of MO (medulla ablongata): dorsal side of soft water, Anterior direction on anterior-posterior axis: anterior of the anterior-posterior axis Direction, superior direction on superior-inferior axis: upper direction of upper-lower axis, right direction on right-left axis: right direction of left-right axis]
4 is a diagram showing an analysis of a blood vessel pattern in a cranial nerve. White arrows indicate porus acusticus.
[Nasal vault: Nasal vault, Olfactory nerve: Olfactory nerve, Vascularity of O.nerve: Vascularity of the olfactory nerve, IAC (internal auditory canal): Internal auditory canal, Anterior direction on anterior-posterior axis: Anterior direction on anterior-posterior axis, superior direction on superior-inferior axis: right direction on right-left axis: right direction on left-right axis]
5 is a diagram showing the distribution of blood vessels in the spinal cord. The black asterisk represents the spinal cord, and the white arrow represents the spinal cord branch.
[SC(spinal cord): spinal cord, Ventrum of sc: ventral side of spinal cord, Dorsal portion of sc: dorsal side of spinal cord, A of spinal branches: spinal branch artery, Sacral A: sacral artery, Anterior direction on anterior-posterior axis: anterior -Forward direction of rear axis, superior direction on superior-inferior axis: upper direction of upper-lower axis, right direction on right-left axis: right direction of left-right axis]
6 is a view showing the structure of the Willis ring of 6 kinds of experimental animals and comparing the branching angles of the Willis ring, FIGS. 6A to F show the Willis ring of 6 kinds of experimental animals and the distribution of blood vessels around it, and FIG. 6G is The distribution of blood vessels after removal of the Willis ring of 6 kinds of experimental animals and the vascular branches around them is shown, FIG. 6H shows the posterior transit artery of Gerville, and 6I shows the angles of each branch of the Willis ring of 6 kinds of experimental animals, Figure 6J shows the anterior intake through the carotid artery hole of the guinea pig, Figure 6K shows the dominance of the right posterior traffic artery of the hamster.
[Anterior cerebral A: Anterior cerebral artery, Posterior communicating A: Posterior transport artery, Inserting portion of internal carotid A: Anterior direction on anterior-posterior axis: superior direction on superior-inferior axis: right direction on right-left axis: right direction on left-right axis]
7 is a view showing the structure of the basal artery of six types of experimental animals, and a comparison of the branches and branching points of the basal arteries, and FIGS. 7G shows the distribution of blood vessels after removal of the cerebral basal arteries and the vascular branches around them of 6 kinds of experimental animals, FIG. 7H is a diagram showing the inflow of the vertebral arteries into the brain, and FIG. 7I is a single anterior spinal artery of a guinea pig. 7J is a view showing the dorsal side of the hamster's soft water, and FIG. 7K shows the angles of each branch of the basal artery of the six types of experimental animals.
[AICA: anterior cerebellar artery, PICA: posterior inferior cerebellar artery, Anterior spinal A: anterior spinal artery, Pontine A: cerebral artery, Anterior direction on anterior-posterior axis: superior direction on superior-inferior axis: Upward direction of the upper-lower axis, right direction on right-left axis: Right direction of the left-right axis]
Figure 8 is a diagram showing the vascular distribution pattern of olfactory cells of six kinds of experimental animals and the analysis method of the blood vessel distribution pattern of the brain parenchyma, and the pituitary vascular distribution, and Figs. 8A to D are the olfactory bulbs of mice, gerbils, hamsters and guinea pigs. bulb) (red line: main supply arterioles, yellow line: connecting arterioles, dark blue line: adjacent arteries, light blue line: connecting arterioles derived from adjacent arteries), Figure 8E is the cerebral cortex of guinea pigs Figure 8F is a diagram showing the result of staining the pituitary gland of a hamster, Figure 8G is a diagram showing the blood supply to the pituitary gland of Gerbil, Figure 8H is a diagram showing the blood supply to the pituitary gland of KWM, Figure 8I is a Gerbil It is a diagram showing the blood supply of the pituitary gland.
[Anterior direction on anterior-posterior axis: anterior direction of the anterior-posterior axis, superior direction on superior-inferior axis: the upper direction of the upper-lower axis, right direction on right-left axis: the right direction of the left-right axis]

Hereinafter, the present invention will be described in more detail through the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1: Description of experimental animals

In the examples and experimental examples of the present application, the following six kinds of animals were used.

1) Korean wild mouse (KWM); Male, 8 weeks old, 10.8g ~ 13.2g, n=3 (Laboratory Animal Resources Center, Hallym University, Chuncheon-si, Gangwon-do, Korea),

2) C57BL/6J mouse; Male, 9 weeks old, 20.7g ~ 22.1g, n=3 (DBL Co., Eumseong-gun, Chungcheongbuk-do, Korea),

3) F344 rat; Male, 9 weeks old, 199.8g ~ 231.9g, n=3 (SLC, Hamamatsu, Shizuoka Prefecture, Japan)

4) Mongolian gerbil; Male, 10-12 weeks old, 57.4g ~ 61.3g n=3 (Laboratory Animal Resources Center, Hallym University, Chuncheon-si, Gangwon-do, Korea),

5) Syrian hamsters; Male, 10 weeks old, 128g ~ 143.5g n=3 (SLC, Hamamatsu, Shizuoka Prefecture, Japan),

6) Guinea pigs; Male, 9 weeks old, 376.8g ~ 424.7g n=3 (SLC, Hamamatsu, Shizuoka Prefecture, Japan).

The animals were kept in a facility maintained under standard conditions and a consistent environment (temperature 22 ± 2° C., relative humidity 55 ± 10%, 12 hours light, 12 hours dark cycle), and normal rodent pellet feed and water were provided at random. Experiments using the animals were performed according to the guidelines of the Animal Experimental Ethics Committee of Hallym University (Hallim 2017-28).

Example 2: staining of experimental animals

To confirm the vascular anatomy of the animal of Example 1 _{, the following method was used using Alcian Blue 8GX: C 56} H ₆₈ Cl ₄ CuN ₁₆ S ₄ , MW: 1,298.86; Sigma, Saint Louis, Missouri The tissue was stained with. Specifically, the animals were anesthetized with 4% isopluran mixed with oxygen using an anesthesia device for rodents (RC2-Rodent Circuit Controller, VetEquip, Inc., Pleasanton, CA, USA). After the chest wall of the animal was removed after anesthesia, the exposed heart was fixed with forceps and a 26 gauge injection was inserted into the left ventricular tip. For blood removal and initial fixation of animal tissue, saline and 4% paraformaldehyde (PFA) were perfused through the left ventricle at 55 rpm for 5 minutes using a pump system (Watson Marlow 302S Peristaltic Pump, Watson-Marlow, Cornwall, England). Made it. After fixing the animal tissues including the brain and circulatory system, a 0.2% Alcian Blue solution was circulated through the animal's vascular system using a perfusion pump system. The 0.2% Alcian Blue solution was prepared containing 4% paraformaldehyde, and the following amounts were used for each experimental animal; KWM 20ml, Mouse 20ml, Rat 40ml, Mongolian Gerbil 40ml, Syrian Hamster 40ml, Guinea Pig 80ml.

After staining, the arterial system and highly vascularized organs appeared light blue (FIG. 1). Alcian blue, with a molecular weight of 1,340 Da, did not diffuse to the brain parenchyma and spinal cord. Since the blood-brain barrier (BBB) has a blocking range of 400-600 Da, the staining process was limited to the outside of the BBB, and organs with highly developed blood vessels such as liver, kidneys and muscles were also stained.

In addition, in order to understand the anatomy in more detail, another bio-staining agent, Trypan blue reagent (Cat. N. T6146, Sigma, Saintlouis, Missouri) was used, and saline solution and trypan were used in a similar manner to the above. The blue reagents were combined and stained by cycling for 5 minutes using a perfusion pump system. The following concentrations of trypan blue were used for each experimental animal; KWM 0.05%, mouse 0.05%, rat 0.1%, Mongolian gerbil 0.1%, Syrian hamster 0.1%, guinea pig 0.1%.

Example 3: Dissection of experimental animals

In Example 2, the skin was removed from the tip of the nose to the level of the shoulder in order to observe the blood vessels of the central nervous system of the animal body that had been stained in Example 2, and the animal was dissected in the following manner to observe the entire contour of the sample. Specifically, first, the Lambdoid suture line was fractured using a curved metzenbaum, and the parietal and frontal bones were removed. Next, the occipital bone was removed and the incision was extended to the coccyx area (all vertebral fragments were removed). The entire dorsal side of the central nervous system was identified and photographed (x6.7-x50, Nikon stereoscope, SMZ 745 T, and Nikon eXcope T300), and all sides of the dorsal brain and spine were identified. The olfactory nerve was observed after the nasal ceiling was removed.

Next, the brain and spinal cord were removed from the skull base and vertebrae (FIG. 1). To do this, the coccyx area was initially dissected. The spine was cut from the coccyx area to the cervix using a surgical microscope (ZEISS OPMI 1FC) and fine scissors. Next, several blood vessels (vertebral arteries [VAs], common carotid arteries [CCAs]) and cranial nerves were carefully incised using fine scissors, and the spinal cord and the abdominal part of the brain were identified. Next, the cochlea, facial and vestibular nerves were evaluated. Finally, the inner surfaces of the cranial base and vertebrae were confirmed (FIG. 1).

Example 4: Central nervous system image acquisition

In order to obtain the clearest image, the objective lens of the stereo microscope and the specimen were placed in parallel (to observe three-dimensionally and to check the exact length and area of the structure). Illustrations of the cerebral artery ring (Circle of Willis, CoW) and basilar artery (BA) were produced, and the images were analyzed using Image J software (https://imagej.nih.gov) ( Fig. 1, middle image).

Example 5: Statistical treatment

For each species, to compare the vascular branching angles of the two animals, the Mann-Whitney test was used. The Kruskal-Wallis Test was not used because of multiple comparison problems and type 1 errors in statistics. Statistical analysis was performed using SPSS version 21 (SPSS IBM, USA), and the significance level was set to p <0.05.

Experimental Example 1: Observation of blood vessel distribution (blood system) in the brain of experimental animals

After staining and dissecting the experimental animals in the above example, the blood vessel distribution in the brain was observed. First, with regard to the brain's lower circulation (VA to CoW), all experimental models (n=3 per animal) all showed excellent staining results. Specifically, it was observed that CoW surrounds the hypothalamus in all species (Figs. 2A-F), and branches of BA differed between species (Fig. 2G-L). In some animals, a single pons artery was observed (Figs. 2B, E, F). Also, at the maximum magnification (x50), the nuclei of the main blood vessels (the first branch of BA and BA) were observed.

Next, the bifurcation and anastomosis patterns of the anterior cerebral arteries (ACA) were observed differently for each species (Fig. 2M-R). In B6 mice, gerbils and hamsters, double ACA (double arterial deformity in the form of a button pupil) was observed (Fig. 2N-P), and a single ACA was observed in the remaining species. Anterior communicating artery was not observed in all species. The small arterioles (gastric pituitary arteries) attached to the primary capillary plexus of the hypophyseal portal system (HPS) are shown in the arrow in Fig. 2P. The shape of HPS was different for each species (Fig. 2S-X), and the HPS of rodents was different from that of humans. In addition, the vascular distribution at the base of the skull was well observed in all species (Fig. 2Y-d).

Regarding the vascular distribution of the soft water, cerebral and cerebellum, it was observed that the branched arteries from BA were anastomized with each other in a reticulated pattern on the soft water (Figs. 3A-F). It was observed that the cerebellum around the fissure and the dorsal-anterior region of the cerebral parenchyma were supplied from the branch of the meningeal artery in the triangular region (Fig. 3G-L). This meningeal artery is derived from the pterygoid artery (spine artery) derived from the internal carotid artery. The branches of the middle cerebral arteries (MCA) were different for each species, and the vascular distribution of soft water is shown in FIGS. 3S-X.

Experimental Example 2: Observation of blood vessel distribution (vascular system) of the cranial nerve

After staining and dissecting the experimental animals in the above example, the vascular distribution of the cranial nerves (the olfactory nerves and the nerves in the internal auditory canals) was observed. Specifically, the vascular supply to the olfactory nerve was visualized as a single artery supplying the olfactory nerve and a small artery supplying the lateral portion of the olfactory nerve derived from the olfactory bulb region (FIGS. 4A-F). In guinea pigs, a single nerve parenchyma such as a ganglion was observed between the olfactory bulb and the olfactory nerve (Fig. 4F, white arrow). After removing the nasal ceiling, it was observed that the nerve bundles were located on the ethmoid bone according to the shape of each animal (FIG. 4G-L), and the blood vessel patterns were different from each other (FIG. 4M-R). Regarding the vascular distribution of the internal ear canal (facial, cochlear, vestibular nerve), it was observed that the nerves of the internal ear canal are supplied from various arteries (FIGS. 4S-X). In the case of the rat brain, a distinct artery was clearly shown for each nerve (FIG. 4W). In addition, it was observed that hamsters, rats and guinea pigs have separate pores for the cochlea, facial and vestibular nerves (Figs. 4V-X).

Experimental Example 3: Observation of blood vessel distribution (vascular system) of the spinal cord

After staining and dissecting the experimental animals in the above example, the vascular distribution of the spinal cord was observed. Specifically, an anterior spinal artery was observed in the ventral side of the spinal cord (FIGS. 5A-F). The anastomosis of the medullary artery was several times over the length of the anterior spinal artery. In the case of mice, gerbils and hamsters, it was observed that the anterior spinal artery deviated toward the anterior medullary artery (FIGS. 5B-D), but maintained in a straight line in other animals (FIGS. 5A, E, F). The dorsal artery of the spinal cord is shown in Fig. 5G-L, and the vascular distribution of the muscle artery to the spinal cord branch is shown in Fig. 5M-R. Single or multiple arterioles were supplied to the spinal cord branches in species other than rats. It was observed that the rat had a network of arteries formed from the spinal meninges (FIG. 5Q), and from this, it was observed that the fused blood vessels to form several arterioles were supplied to the spinal cord branch (FIG. 5Q). In the sacral region, it was observed that a single dominant artery supplies the vertebral branch (Fig. 5S-X).

Experimental Example 4: Observation of the angle of the Circle of Willis (CoW)

After staining and dissecting the experimental animals in the above example, the Circle of Willis (CoW) was observed. First, in order to understand the Cow observation result, the acquired image was processed "underpainting 1" ( www.befunky.com ), and the image was converted by inverting (negative) (FIGS. 6A-F). As a result, the inverted image of the artery stained with Alcian Blue could be converted to the actual blood vessel color. However, staining of small arterioles and surrounding tissues provided inaccurate images when compared with the actual in vivo structure. Therefore, unnecessary red areas were removed after cartooning (cartoon correction) using image software. As a result, all images were successfully converted into virtual real-images (Fig. 6G).

As a result of analyzing the images obtained above, it was observed that the posterior cerebral artery (PCA) of six experimental animals had a unique shape. Specifically, in most of the experimental animals except KWM (wild mouse), hexagonal CoW was observed, PCA was not observed only in KWM (Fig. 6G), and in the case of Gerbil, a multi-vascular network was observed between CCA and BA ( 6G, H), in the case of guinea pigs, a trophy shape was shown due to the unique CCA location (FIGS. 6G, J). In the case of hamsters, PCA branched from BA at an obtuse angle (Fig. 6G). In general, BA is known to supply blood to the occipital brain (mainly the pons and cerebellum). In most animals, the same results were observed, but in the case of hamsters, the contour of the distal portion of the BA is judged to be the dominant connective artery from the BA as correct PCA (Fig. 6K), but it seems that additional studies are needed to confirm this. .

Next, the angle branching from BA was calculated forward (Fig. 6I). The branching angle of BA is important in determining the cerebral vascular distribution or hemodynamic analysis of ischemia. In addition, with regard to the connection angle of the ACA, the compensatory anastomosis (anterior traction artery) is important after cerebral infarction. As a result of observation in six experimental animals, an acute angle was observed in the case of guinea pigs, but a relatively wide angle was observed in the case of other animals (FIG. 6I).

Based on the above results, by using the method of the present application, it is possible to check the structure of the Willis ring artery and the angle of the branch, and it is possible to efficiently check the Willis ring artery abnormality, and based on this, a disease caused by an abnormality of the Willis ring artery. Can be diagnosed.

Experimental Example 5: Basilar artery (BA) observation

After staining and dissecting the experimental animals in the above example, the basilar artery (BA) was observed. Images obtained from six experimental animals were analyzed after processing the images through image reversal and cartoon correction (FIGS. 7A-G), and as a result of observation, branching patterns were different for each species. Specifically, it was observed that a double feed (hamster, image below Fig. 7H) or a single artery (guinea pig, Fig. 7I) extends toward the abdomen of the spine. In addition, it was observed that the posterior spinal artery is derived from the branch of the posterior inferior cerebellar artery (PICA), and the two branches from PICA merge into an arterial sling, and the branch is radially spread. (Fig. 7J black arrow). The radially spreading branch of the main medial artery is the posterior vertebral artery (Fig. 7J). The summation angles for both VA and anterior inferior cerebellar artery (AICA) were different for each species, and were not statistically significant (FIG. 7K).

Based on the above results, by using the method of the present application, it is possible to check the structure of the cerebral basal artery and the angle of the branch, and it is possible to efficiently check the cerebral basal artery abnormality, and based on this, a disease caused by an abnormality of the cerebral basal artery. Can be diagnosed.

Experimental Example 6: Observation of circulation of olfactory nerves, brain parenchyma and pituitary gland

Although the sense of smell is the most important for the survival of rodents, it is considered that the functional characteristics of the sense of smell will vary in inbred experimental animals. In the case of KWM, since the vascular system of the olfactory nerve was not stained, differences between wild animals and inbred animals could not be compared. In the following, experiments were conducted on mice, gerbils, hamsters, and guinea pigs that can confirm the vascular supply pattern of the olfactory nerve (FIGS. 8A-D).

For comparison, the main branch was observed by comparing the straightness and the width (Fig. 8A-D red line), and the connecting branch was separately indicated (Fig. 8A-D, yellow line). As a result, in the case of the mouse, a diffused branching pattern was observed, in the case of a gerbil, an unconnected stripe pattern was observed, and in the case of a hamster, a diffused branching and merging pattern were observed. In the case of guinea pigs, feeding arterioles derived from ethmoid arteries were observed (Fig. 8D, blue line).

Since the brain parenchyma has a blood-brain barrier (BBB), the dye could not reach the brain parenchyma directly. As a result, it was not difficult to identify the vascular system in the brain. Specifically, in the case of guinea pigs, the number of branches of the middle cerebral artery (MCA) on the cerebral surface was confirmed and counted (FIG. 8E). After the 5th branch point, the small arterioles turned 90 degrees toward the parenchyma, and anastomosis with the medial supply artery was observed (Fig. 8E, right image). In contrast, in rats, arterial anastomosis was observed at all levels of branches from the main artery to the capillaries.

The pituitary gland can be divided into an anterior pituitary gland and a neuro pituitary gland, and both are endovenous periphery organs, which means having a highly permeable microvascular system (FIG. 8 F-1, F-2, G). In the case of hamsters, several arterioles from CoW are observed (Fig. 8F-2). Weak staining means that the arteries and arterioles are stained using a dye that is out of the pituitary parenchyma (Figs. 8F-3 to F-5). However, strong staining means complete staining of the pituitary gland with a strong supply to the arteries (Figs. 8F-6 to F-8). The red color in Figs. 8F-9 and F-10 indicates the remaining blood due to the venous circulation disorder in the neuro pituitary gland (white arrow in Fig. 8F-11). From this result, it can be inferred that the circulation of the pituitary gland and the anterior pituitary gland were separated. In addition, it can be seen that the pituitary gland is supplied from several supply arterioles derived from CoW, and the anterior pituitary gland is supplied from a single arteriole in which several branched arterioles of CoW are anastomized (FIG. 8F-12). In addition, the above results show different results for each animal. Despite staining in the same way, the spherical pituitary gland of KWM was stained as shown in Figs. 8F-6 to F-8 (Fig. 8H), and the pituitary gland of Gerville's octagonal shape was stained as shown in Figs. 8F-9 to F-11. (Fig. 8I). Taking the above results together, it can be seen that the microvascular system has different permeability depending on the species and individual of the animal.

From the above description, those skilled in the art to which the present application pertains will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the examples and experimental examples described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including the meaning and scope of the claims to be described later rather than the above detailed description, and all changes or modifications derived from the equivalent concepts included in the scope of the present invention.

Claims (11)

(a) injecting a 0.2% Alcian Blue solution containing 4% paraformaldehyde into the blood vessel of the subject; And
(b) circulating the 0.2% Alcian Blue solution containing 4% paraformaldehyde to stain the blood vessels and surrounding tissues of the individual;
Containing, central nervous system blood vessel staining method of animals other than humans.
delete delete delete The method of claim 1,
The central nervous system blood vessels are Willis-hwan (Circle of Wills, CoW) arteries and the basal artery (basilar artery, BA) containing the central nervous system blood vessel staining method of animals other than humans.
(a) injecting a 0.2% Alcian Blue solution containing 4% paraformaldehyde into the blood vessel of the subject;
(b) circulating the 0.2% Alcian Blue solution containing 4% paraformaldehyde to stain the blood vessels and surrounding tissues of the individual;
(c) obtaining an image of the stained brain and spinal cord; And
(e) converting the obtained image
Comprising a method for visualization of blood vessels of the central nervous system of animals other than humans.
The method of claim 6,
The image conversion is to include at least one selected from the group consisting of color inversion imaging and cartoon imaging, the method for visualizing the central nervous system blood vessels of animals other than humans.
(a) visualizing the blood vessels of the central nervous system of the individual using the method according to claim 6; And
(b) confirming the mutation of the visualized central nervous system blood vessels
Containing, a method of providing information for diagnosing cerebrovascular disease.
The method of claim 8,
The cerebrovascular disease is selected from the group consisting of cerebral aneurysm, cerebral infarction, cerebral thrombosis, cerebral embolism, cerebral hemorrhage, and stroke.
(a) administering a candidate material for cerebrovascular disease treatment to an animal model of cerebrovascular disease; And
(b) visualizing the blood vessels of the central nervous system of the animal model of cerebrovascular disease to which the candidate substance was administered using the method according to claim 6; And
(c) confirming the degree of variation in blood vessels of the central nervous system according to the administration of the candidate substance.
Containing, a method for screening a therapeutic agent for cerebrovascular disease.
The method of claim 10,
The method further comprises determining the candidate substance as a treatment for cerebrovascular disease when the degree of variability in the blood vessels of the central nervous system is alleviated according to the candidate substance for treatment of cerebrovascular disease.

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