WO2020159264A1 - Method for evaluating synaptic dysfunction using brain tissue clearing - Google Patents

Method for evaluating synaptic dysfunction using brain tissue clearing Download PDF

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WO2020159264A1
WO2020159264A1 PCT/KR2020/001442 KR2020001442W WO2020159264A1 WO 2020159264 A1 WO2020159264 A1 WO 2020159264A1 KR 2020001442 W KR2020001442 W KR 2020001442W WO 2020159264 A1 WO2020159264 A1 WO 2020159264A1
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solution
brain tissue
concentration
sample
synaptic dysfunction
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박영일
금상일
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주식회사 바이나리
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4076—Diagnosing or monitoring particular conditions of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons

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  • the present invention relates to a method for evaluating synaptic dysfunction using transparent brain tissue.
  • neurological diseases including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, Alzheimer's, diabetic retinopathy, multiple infarction dementia, and discoid macular degeneration is also increasing. It is estimated that there are currently 24 million people with neurological disorders worldwide. Also, diseases caused by stroke or other trauma or damage, which are other types of nervous system diseases, are on the increase every year.
  • neurological disorders still suffer from this disorder despite advances in diagnosis and treatment, and neuronal damage can further exacerbate the development of neurological disorders such as epilepsy, resulting in an imbalance of excitement and inhibition, including brain dysfunction.
  • structural and functional defects of spine present in dendrites of neurons appear as the cause or result of a number of nervous system diseases, and thus determine brain activity including synapse formation and synaptic plasticity as a pathway and starting point for neurotransmission. Studying the structure and characteristic features of the spine in the characteristic structural minimum unit is an essential process for understanding the spine and nervous system formation, and also the cognitive action of the brain.
  • tissue transparent technology can confirm the structure and protein expression without damaging the tissue
  • a technology capable of transparent tissue is developed in a variety of ways.
  • Existing tissue clearing techniques have been reported for the antigen preservation of tissues treated by Spatleholz, BABB, Scale S, iDISCO, which is a method for tissue clearing using organic solvents, and ACT (active CLARITY technology), which is a polymer injection method.
  • ACT active CLARITY technology
  • fluorescence and antigen retention are reduced.
  • ACT it has an antigen preservation of 90% or more, and it shows a higher preservation property compared to a method requiring binding to a hydrogel polymer in addition to a fixed protein such as CLARITY.
  • a strong tissue fixation process causes loss of antigenicity, so problems such as reduction of usable antibodies must be considered, and thus, various techniques need to be improved.
  • the recently developed CLARITY method uses a method to selectively remove lipids after making a kind of mesh support that holds a material such as DNA or protein by inserting a hydrogel in tissue to hold important materials for diagnosis.
  • Tissue transparency technologies that can create optically transparent and polymer-transmissive images, such as CLARITY and perfusion aided reagent release methods (PARS), have provided major advances in highly enhanced organ system imaging.
  • the clarity method has a disadvantage in that, as the hydrogel concentration increases, the degree of binding to the protein increases and the tissue becomes harder and thus the removal of lipids becomes difficult. This causes deposition of air and black particles on the tissue surface.
  • the existing Clarity method is a complicated process and requires a lot of additional equipment.
  • only one tissue can be transparent at a time, causing economic and time loss, and staining with antibodies has been unstable.
  • the present inventors do not require an expensive electrophoresis device and transparent the entire brain tissue using a clearing solution capable of increasing the transparency of biological tissue without damaging various tissues, and analyze the neurons in the brain tissue to analyze the nervous system It was intended to invent a method for evaluating synaptic dysfunction that can be used for disease-related research.
  • the present inventors prepare a clearing solution capable of increasing the transparency of biological tissues without damaging various tissues without the need for expensive electrophoresis devices, and using this to clear the brain tissue to obtain a clear 3D fluorescence image of neurons Invented a method of evaluating synaptic dysfunction by analyzing the length and distribution of the neurons.
  • an object of the present invention is to provide a method for evaluating synaptic dysfunction, comprising the following steps:
  • Another object of the present invention is to provide a method for screening candidates for preventing or treating neurological diseases, comprising the following steps:
  • the present invention provides a method for evaluating synaptic dysfunction, comprising the following steps:
  • the present invention provides a method of screening a candidate substance for preventing or treating a nervous system disease, comprising the following steps:
  • the fixed solution may include sucrose.
  • the sucrose may have a concentration of 20% (w/v) to 100% (w/v).
  • the tissue clearing solution is N-Lauroylsarcosine sodium salt solution, CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) , Urea (urea), and sodium chloride (NaCl).
  • the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v), and CHAPS ( 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/v), and the urea has a concentration. 30% (w/v) to 70% (w/v), and the sodium chloride (NaCl) concentration may be 0.001% (w/v) to 1% (w/v).
  • the washing solution may include phosphate buffer saline (PBS) and sodium azide.
  • PBS phosphate buffer saline
  • the sodium azide may have a concentration of 0.001% (w/v) to 0.5% (w/v).
  • the mounting solution is composed of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include one or more selected from the group.
  • the concentration of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/ v)
  • the concentration of urea (urea) is 10% (w/v) to 50% (w/v)
  • the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3%. .
  • step (e) may be using an Imaris program.
  • the step of clearing the brain tissue of the animal may include the following steps:
  • the method for evaluating synaptic dysfunction according to the present invention includes the step of making the brain tissue transparent, and by clearing the brain tissue in the above step, a clear three-dimensional image of the neuron can be obtained, and the neurons of the neurons through the Imaris program. Synaptic dysfunction can be assessed by effectively analyzing and quantifying spine length and distribution.
  • the method for evaluating synaptic dysfunction of the present invention is expected to be useful for screening drugs for preventing or treating neurological diseases by analyzing the spine length and distribution of neurons according to the treatment of drugs in animal models of neurological diseases. do.
  • FIG. 1 is a view showing a three-dimensional fluorescence image of neurons obtained by clearing and immunostaining brain tissue using a clearing solution according to one embodiment of the present invention.
  • FIG. 2 is a diagram comparing a 3D fluorescent image of neurons obtained when the brain tissue is transparent with the transparent method according to an embodiment of the present invention, compared with the image obtained when the brain tissue is transparent with the CLARITY method.
  • FIG 3 is a view showing the results of observing the spine length and distribution of neurons through the Imaris program, and transparent the brain tissue using a clearing solution according to an embodiment of the present invention.
  • the present invention provides a method for evaluating synaptic dysfunction, comprising the following steps:
  • the present invention provides a method of screening a candidate substance for preventing or treating a nervous system disease, comprising the following steps:
  • neuron is a unit of the nervous system, also called a neuron, and can be electrically excited, and converts information into a chemical or electrical signal and transmits it.
  • Signal transmission between neurons occurs through specialized connections called synapses, and several neurons are connected to each other to form a neural network.
  • neurons extend from neuronal cell bodies (cell bodies, soma), which contain large, round nuclei, and various organelles necessary to maintain the function of cells, and receive information from other neurons. It consists of a dendrite, and an axon used by neurons to transmit an electrical signal called an active voltage.
  • dendritic spine used in the present invention is used in the same sense as “dendritic spine", and the dendritic spine is a small protruding structure of 0.5-2 ⁇ m length present on the surface of the neuronal dendrite. As, it functions as a post-synaptic synapse.
  • the number, size, and shape of dendritic spines present in neurons vary widely and vary depending on the development process of neurons and the degree of activity of individual synapses.
  • Various neurotransmitter receptors required for postsynaptic function are located on the dendritic spine surface, and there are hundreds of different types of proteins required for molecular signaling and control of the dendritic spine structure.
  • synapte refers to a region of a neuron through which the neuron passes electrical or chemical signals to another cell.
  • the plasma membrane of signal-passing neurons is almost identical to the membrane of the target (post-synaptic) cell.
  • the candidate substance refers to an unknown substance used for screening to confirm the efficacy of preventing or treating a nervous system disease by analyzing the spine length and distribution of the neurons in brain tissue of an animal model of a nervous system disease,
  • a chemical, protein, (poly) nucleotide, antisense-RNA, siRNA (small interference RNA), or natural extracts may be included, but are not limited thereto.
  • the candidate material may be a therapeutic agent for a nervous system disease.
  • nerve system disease causes loss of nerve function by the death or degeneration of nerve cells, such as brain cells, temporarily or over a long period of time, thereby causing cognitive, sensory, motor, and systemic functions.
  • nerve cells such as brain cells
  • cognitive, sensory, motor, and systemic functions Refers to the condition or symptom of deterioration, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick disease, Krofelz- Creutzfeld-Jacob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, cortical basal degeneration, multiple system atrophy Multiple system atrophy, progressive supranuclearpalsy, neurological autoimmunedisease, multiple sclerosis, inflammatory and neuropathic pain and neuro vascular disease disease) may be any one or more selected from the group consisting of, but is not limited to the type of the nervous system disease.
  • the step of clearing the brain tissue of the animal may include the following steps:
  • the animal is not particularly limited in its kind, and may be, for example, a human, non-human primate, mouse, dog, cat, rabbit, horse, or cow.
  • the animal may be a mouse, and a brain tissue image of the mouse may be better observed using a Thy-1 transgenic mouse over-expressing Thy-1, but is not limited thereto.
  • the fixed solution may include sucrose (sucrose), the sucrose (sucrose) has a concentration of 20% (w/v) to 100% (w/v), 20% (w/v) To 70% (w/v), 20% (w/v) to 30% (w/v), 30% (w/v) to 40% (w/v), 40% (w/v) to 50 %(w/v), 50%(w/v) to 60%(w/v), 60%(w/v) to 70%(w/v), 70%(w/v) to 80%( w/v), or 80% (w/v) to 100% (w/v).
  • sucrose sucrose
  • sucrose sucrose
  • the sucrose (sucrose) has a concentration of 20% (w/v) to 100% (w/v), 20% (w/v) To 70% (w/v), 20% (w/v) to 30% (w/v), 30% (w/v) to 40% (w/v), 40% (w/v) to 50 %(w/v), 50%(w/v) to 60%(
  • the sample may be dehydrated by setting the concentration of sucrose to 20% (w/v) or more, and the sample covalently bound between organic substances with PFA (paraformaldehyde) may be more strongly fixed, but limited to the concentration. It does not work.
  • PFA paraformaldehyde
  • the tissue clearing solution is N-Lauroyl sarcosine sodium salt solution (N-Lauroylsarcosine sodium salt solution), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane Sulfonate), urea (urea), and sodium chloride (NaCl).
  • the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v) or 3% (v/v) to 20. % (v/v), and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/ v) or 10% (w/v) to 30% (w/v), wherein the urea has a concentration of 30% (w/v) to 70% (w/v) or 40% (w) /v) to 60% (w/v), and the concentration of sodium chloride (NaCl) is 0.001% (w/v) to 1% (w/v) or 0.01% (w/v) to 1% ( w/v), and according to a specific embodiment of the present invention, the N-Lauroylsarcosine sodium salt solution is 4% (v/v) or 15% (v/v) ),
  • the step (b) of the clearing using the tissue clearing solution uses a tissue clearing solution containing 4% (v/v) of N-Lauroylsarcosine sodium salt solution.
  • the first clearing step or may include a second clearing step using a tissue clearing solution comprising 15% (v/v) of N-Lauroylsarcosine sodium salt solution.
  • the washing step, the second transparent step, and the washing step may be performed in order, but is not limited thereto.
  • the washing solution may include phosphate buffered saline (PBS) and sodium azide
  • the sodium azide has a concentration of 0.001% (w /v) to 0.5% (w/v) or 0.01% (w/v) to 0.5% (w/v)
  • the sodium azide is After clearing the concentration to 0.1% (w/v), after increasing the water content up to 30% in a dehydrated biotissue sample, dehydrating 15% to wash the organic matter that interferes with imaging attached to the tissue, but the concentration It is not limited to.
  • the mounting solution is one selected from the group consisting of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include the above.
  • the concentration of the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/v) or 30% ( w/v) to 50% (w/v), and the urea has a concentration of 10% (w/v) to 50% (w/v) or 20% (w/v) to 50%.
  • the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3% or 0.01% (w/v) to 2%, according to a specific embodiment of the present invention
  • the composition of the CHAPS and urea may include 40% (w/v) and 40% (w/v), respectively, and the concentration of sodium chloride (NaCl) is 0.1 to 1%. (w/v), but is not limited to the concentration.
  • step (e) may be using an Imaris program.
  • a solution for clearing the brain tissue of a Thy-1 transgenic mouse is prepared, and the brain tissue of the Thy-1 transgenic mouse is cleared using the solution, followed by immunostaining, to provide three-dimensional immunity of neurons. Dyed images were obtained (see Example 1).
  • the spine length and distribution of neurons were observed in the transparent brain tissue using the Imaris program, and it was expected to be able to confirm whether or not synaptic dysfunction occurred. (See Example 2).
  • a fixation solution a tissue clearing solution, a washing solution, and a mounting solution required for tissue clearing were prepared, and the components of the solution are shown in Table 1 below.
  • a fixed solution containing sucrose having a concentration of 20% (w/v) or more as a component a fixed solution containing sucrose having a concentration of 20% (w/v) or more as a component
  • N-Lauroylsarcosine sodium salt at a concentration of 4% (v/v) or 15% (v/v) to minimize the degeneration of the fluorescent substance in the biological tissue sample by stabilizing the tissue deformation and ion strength by osmotic pressure.
  • solution 0.1% to 0.5% in 20% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 50% (w/v) urea
  • NaCl sodium chloride
  • PBS phosphate buffer saline
  • Configuration Composition One Fixing solution Sucrose (more than 20% (w/v)) 2 Tissue clearing solution N-Lauroylsarcosine sodium salt solution (4% (v/v) or 15% (v/v)), CHAPS (20% (w/v)), Urea (50% (w/v)), NaCl (0.1 to 0.5% (w/v)) 3 Washing solution PBS, sodium azied (0.1% (w/v)) 4 Mounting solution CHAPS (40% (w/v)), Urea (40% (w/v)), NaCl (0.1 to 1% (w/v))
  • a brain tissue sample of Thy-1 GFP transgenic mouse was clarified using the solution prepared in Example 1-1 (see Table 1).
  • Thy-1 GFP transgenic mouse was purchased from The Jackson Laboratory, and inhaled anesthesia using isoflurane as an anesthetic gas to obtain a brain sample of the mouse, and mixed with 100% oxygen.
  • the cells were opened and cardiac perfusion was performed with 30 ml PBS (pH 7.2) for 3-5 minutes, followed by perfusion with 30-50 ml of 4% paraformaldehyde for 20-30 minutes to obtain brain tissue samples of mice.
  • the brain tissue sample of the mouse obtained from the above was placed in a fixing solution, shaking incubation until precipitation at 4°C/50 rpm, and the precipitated brain tissue sample was 4% (v/v) N- It was added to a tissue clearing solution containing lauroylsarcosine sodium salt solution, shaking incubation for 36 hours at 35°C/50 rpm, and repeated once under the same conditions.
  • the washed brain tissue sample was placed in a mounting solution, shaking incubation at 35°C/50 rpm for 12 hours, and repeated once under the same conditions.
  • the brain tissue sample treated with the mounting solution was centrifuge for 30 minutes at 1200 rpm to remove bubbles in the sample, and the brain tissue sample was stored in an image chamber or stored at 4°C in 1X PBS.
  • the fluorescence amount of Thy-1 GFP of the brain tissue, which had been cleared by the above method, was measured using a lightsheet microscope. As a result, as shown in FIG. 1, it was possible to confirm a three-dimensional fluorescence image of clear neurons in the brain tissue transparent by the above method.
  • Example 1-2 After the brain tissue was made transparent by the method of Example 1-2, the spine length and distribution of neurons were observed in a fluorescence image of neurons in three dimensions using an Imaris 3D program.
  • the neuron cell body and dendrite of the neuron were separated.
  • the selection area from the starting point (set the diameter of the dendrite) to the ending point was checked, the 3D image was matched, and the fluorescence of the image was analyzed to accurately observe the diameter length of the dendrite, and the spine of the dendrite is shown in FIG. 3. As shown, it was confirmed to appear in blue, and the total number, shape, and branching of the spine were analyzed using the settings built into the Imaris program.
  • the dendritic spine is a small protrusion that receives the excitatory signal present in the dendrite of a neuron and has various sizes and shapes.
  • a large spine forms a large synaptic frame proportional to it and has more diverse organelles.
  • Postsynaptic density is a structure near the postsynaptic membrane, usually located on the spine head, occupying about 10% of the surface area of the spine. Since it is proportional to the number of synaptic sacs, the mechanism of spine formation and growth is closely related to the intensity of synaptic signaling.
  • the method for evaluating synaptic dysfunction according to the present invention can be effectively used to identify synaptic dysfunction by effectively analyzing and quantifying the spine length and distribution of neurons, and is also useful for screening drugs for preventing or treating neurological diseases. It is expected to be possible.

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Abstract

The present invention relates to a method for evaluating synaptic dysfunction using brain tissue clearing, and a method for evaluating synaptic dysfunction, according to the present invention, comprises a step of making brain tissue transparent, wherein a clear three-dimensional image of a neuron may be obtained by making the brain tissue transparent, and synaptic dysfunction may be evaluated by effectively analyzing and quantifying the length and distribution of spines of the neuron through the Imaris program. In addition, the method of the present invention for evaluating synaptic dysfunction is expected to be useful for preventing neurological disorders and screening a drug for treatment by analyzing the length and distribution of spines of a neuron according to processing of the drug in animal models of a neurological disorder.

Description

뇌 조직 투명화를 이용한 시냅스 기능 장애 평가 방법 Method for evaluating synaptic dysfunction using brain tissue transparent
본 발명은 뇌 조직 투명화를 이용한 시냅스 기능 장애 평가 방법 등에 관한 것이다.The present invention relates to a method for evaluating synaptic dysfunction using transparent brain tissue.
본 출원은 2019년 02월 01일에 출원된 한국특허출원 제10-2019-0013801호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.This application claims priority based on Korean Patent Application No. 10-2019-0013801 filed on February 01, 2019, and all contents disclosed in the specification and drawings of the application are incorporated in this application.
인간의 수명이 길어짐과 동시에 파킨슨 질환, 근위축성측삭경화증, 다발경화증, 헌팅톤 질환, 알츠하이머, 당뇨망막변증, 다발성경색치매, 원반황반변성 등을 포함하는 신경계 질환의 발생률도 높아지고 있다. 현재 신경계 질환 환자는 전세계적으로 2400만명인 것으로 추정된다. 또한 다른 형태의 신경계 질환인 뇌졸중 또는 기타 외상 또는 손상에 의해 야기되는 질환은 해마다 증가추세에 있다. At the same time as human lifespan is prolonged, the incidence of neurological diseases including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, Alzheimer's, diabetic retinopathy, multiple infarction dementia, and discoid macular degeneration is also increasing. It is estimated that there are currently 24 million people with neurological disorders worldwide. Also, diseases caused by stroke or other trauma or damage, which are other types of nervous system diseases, are on the increase every year.
현재 신경계 질환은 진단 및 치료의 발전에도 불구하고 여전히 이로 인해 장애를 겪고 있는데, 뉴런의 손상은 뇌 기능 장애를 포함하여 흥분과 억제의 불균형을 초래해 간질과 같은 신경계 질환의 발생을 더욱 악화시킬 수 있다. 또한, 뉴런의 수상돌기에 존재하는 spine의 구조적, 기능적 결함은 다수의 신경계 질환의 원인 혹은 결과로 나타나므로, 신경전달의 통로이자 시발점으로써의 시냅스 형성과 시냅스 가소성을 포함하여 뇌의 활동도를 결정하는 특징적인 구조적 최소단위로 spine의 구조와 특징적인 모습들을 연구하는 것은 spine 및 신경계 형성, 나아가 뇌의 인지작용을 이해하기 위해 필수적인 과정이다.Currently, neurological disorders still suffer from this disorder despite advances in diagnosis and treatment, and neuronal damage can further exacerbate the development of neurological disorders such as epilepsy, resulting in an imbalance of excitement and inhibition, including brain dysfunction. have. In addition, structural and functional defects of spine present in dendrites of neurons appear as the cause or result of a number of nervous system diseases, and thus determine brain activity including synapse formation and synaptic plasticity as a pathway and starting point for neurotransmission. Studying the structure and characteristic features of the spine in the characteristic structural minimum unit is an essential process for understanding the spine and nervous system formation, and also the cognitive action of the brain.
임상적으로 신경계 질환의 연구는 뇌 영상 및 전자현미경을 통한 연구, 신경세포의 사망 기전에 대한 분자 생물학적 연구에 힘입어 새로운 변화들이 알려졌다. 또한, MRI 기술의 발전으로 뇌의 위축 등을 쉽게 확인할 수 있으며 전자현미경으로 미세한 시냅스 변화부터 뉴런 사멸의 다양한 기전을 확인할 수 있다. Clinically, the study of neurological diseases is known through molecular imaging and research on brain imaging and electron microscopy, and molecular biological studies on the mechanism of death of neurons. In addition, with the development of MRI technology, it is easy to check the atrophy of the brain, etc., and an electron microscope can confirm various mechanisms of neuron death from minute synaptic changes.
그러나, 전임상에서의 신약 개발 과정에서 뉴런의 손상 정도를 평가할 수 있는 방법은 슬라이드에 조직을 박리하여 관찰하는 방법이 대부분이며, 특히 동물의 전체 뇌조직에서 신경의 분포, 생존, 형태학적인 연구를 위한 시스템 및 분석 방법은 아직까지 잘 알려진 바가 없는 실정이다. However, in the preclinical drug development process, the method of evaluating the degree of damage to neurons is mostly observed by peeling the tissue on the slide, especially for the distribution, survival, and morphological studies of nerves in the entire brain tissue of animals. Systems and analysis methods are not well known.
한편, 조직 투명화 기술은 조직의 손상 없이 구조 및 단백질 발현 등을 확인 할 수 있으므로 최근에 매우 다양한 방법으로 조직을 투명화 할 수 있는 기술이 개발되었다. 기존의 조직 투명화 기술은 유기용매를 이용한 조직 투명화 방법인 Spatleholz, BABB, Scale S, iDISCO법과, 폴리머 주입법인 ACT(active CLARITY technology)법에 의해 처리된 조직의 항원 보존성이 보고된 바 있다. ACT를 제외한 다른 방법의 경우 형광과 항원의 보존성이 감소하는 문제를 가지고 있다. ACT의 경우 90% 이상의 항원 보존성을 가지며, 이는 클라리티(CLARITY)와 같이 고정된 단백질에 추가로 하이드로젤 폴리머와의 결합을 필요로 하는 방법에 비하면 보다 높은 보존성을 보인다. 그러나 강한 조직 고정 과정은 항원성의 손실을 유발하여, 사용할 수 있는 항체가 감소하는 등의 문제점을 고려해야 하므로, 여러 가지 기술의 개선이 필요하다.On the other hand, since the tissue transparent technology can confirm the structure and protein expression without damaging the tissue, recently, a technology capable of transparent tissue is developed in a variety of ways. Existing tissue clearing techniques have been reported for the antigen preservation of tissues treated by Spatleholz, BABB, Scale S, iDISCO, which is a method for tissue clearing using organic solvents, and ACT (active CLARITY technology), which is a polymer injection method. For methods other than ACT, fluorescence and antigen retention are reduced. In the case of ACT, it has an antigen preservation of 90% or more, and it shows a higher preservation property compared to a method requiring binding to a hydrogel polymer in addition to a fixed protein such as CLARITY. However, a strong tissue fixation process causes loss of antigenicity, so problems such as reduction of usable antibodies must be considered, and thus, various techniques need to be improved.
최근에 개발된 클라리티(CLARITY)법은 조직 내 hydrogel을 넣어서 DNA나 단백질 등 진단에 중요한 material 등을 붙잡아 주는 일종의 그물망 지지체를 만든 뒤 지질만을 선택적으로 제거하는 방법을 이용한다. 클라리티(CLARITY) 및 관류 도움 시약 방출방법(PARS)과 같이 광학적으로 투명하고 고분자 투과가 가능한 이미지를 창출할 수 있는 조직투명성 기술은 매우 향상된 기관계 이미징에 있어서 주요한 진전을 제공하였다. The recently developed CLARITY method uses a method to selectively remove lipids after making a kind of mesh support that holds a material such as DNA or protein by inserting a hydrogel in tissue to hold important materials for diagnosis. Tissue transparency technologies that can create optically transparent and polymer-transmissive images, such as CLARITY and perfusion aided reagent release methods (PARS), have provided major advances in highly enhanced organ system imaging.
그러나, 상기 클라리티 방법은 hydrogel 농도가 높아지면 단백질과의 결합도가 많아 더 조직이 단단해져서 지질의 제거가 어려워 지기 때문에 투명화하는데 시간이 오래 걸리는 단점이 있다. 이는 조직 표면에 공기 및 검은 입자의 침착을 야기한다. 또한, 기존의 클라리티 법은 과정이 복잡하고 부가적인 장비가 많이 필요하다. 뿐만 아니라 한번에 한 조직만 투명화 할 수 있어 경제적, 시간적 손실을 유발하며 항체를 이용한 염색이 불안정한 문제가 있었다.However, the clarity method has a disadvantage in that, as the hydrogel concentration increases, the degree of binding to the protein increases and the tissue becomes harder and thus the removal of lipids becomes difficult. This causes deposition of air and black particles on the tissue surface. In addition, the existing Clarity method is a complicated process and requires a lot of additional equipment. In addition, only one tissue can be transparent at a time, causing economic and time loss, and staining with antibodies has been unstable.
이에, 본 발명자들은 고가의 전기영동 장치를 필요로 하지 않고 다양한 조직의 손상 없이 생체 조직의 투명성을 증가시킬 수 있는 투명화 용액을 이용하여 뇌 조직 전체를 투명화하고, 상기 뇌 조직에서 뉴런을 분석하여 신경계 질환 관련 연구 등에 활용할 수 있는 시냅스 기능 장애 평가 방법을 발명하고자 하였다.Accordingly, the present inventors do not require an expensive electrophoresis device and transparent the entire brain tissue using a clearing solution capable of increasing the transparency of biological tissue without damaging various tissues, and analyze the neurons in the brain tissue to analyze the nervous system It was intended to invent a method for evaluating synaptic dysfunction that can be used for disease-related research.
본 발명자들은 고가의 전기영동 장치를 필요로 하지 않고 다양한 조직의 손상 없이 생체 조직의 투명성을 증가시킬 수 있는 투명화 용액을 제조하고, 이를 이용하여 뇌 조직을 투명화 함으로써 선명한 뉴런의 3차원 형광 이미지를 획득하였으며, 상기 뉴런의 길이 및 분포를 분석함으로써 시냅스 기능 장애를 평가하는 방법을 발명하였다.The present inventors prepare a clearing solution capable of increasing the transparency of biological tissues without damaging various tissues without the need for expensive electrophoresis devices, and using this to clear the brain tissue to obtain a clear 3D fluorescence image of neurons Invented a method of evaluating synaptic dysfunction by analyzing the length and distribution of the neurons.
이에, 본 발명의 목적은 하기 단계를 포함하는, 시냅스 기능 장애 평가 방법을 제공하는 것이다:Accordingly, an object of the present invention is to provide a method for evaluating synaptic dysfunction, comprising the following steps:
(a) 고정 용액으로 동물의 뇌 조직 시료를 고정시키는 단계;(a) immobilizing the brain tissue sample of the animal with a fixation solution;
(b) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(b) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
(c) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (c) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
(d) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(d) fixing the washed sample with a mounting solution; And
(e) 상기 (a) 내지 (d) 단계를 거친 뇌 조직 시료에서 뉴런의 스파인(spine) 길이 및 분포를 분석하는 단계.(e) analyzing the spine length and distribution of neurons in a brain tissue sample subjected to steps (a) to (d).
본 발명의 다른 목적은 하기 단계를 포함하는, 신경계 질환 예방 또는 치료용 후보물질의 스크리닝 방법을 제공하는 것이다:Another object of the present invention is to provide a method for screening candidates for preventing or treating neurological diseases, comprising the following steps:
(a) 신경계 질환 동물모델에 후보물질을 처리하는 단계;(a) processing the candidate material in an animal model of a nervous system disease;
(b) 상기 동물의 뇌 조직을 투명화 시키는 단계;(b) making the brain tissue of the animal transparent;
(c) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 뇌 조직에서 뉴런의 스파인(spine) 길이 및 분포를 분석하여 시냅스 기능을 평가하는 단계; 및(c) analyzing synaptic function by analyzing the spine length and distribution of neurons in the cleared brain tissue through the Imaris program; And
(d) 시냅스 기능이 후보물질을 처리하기 전에 비해 개선된 경우 신경계 질환 예방 또는 치료용 후보물질로 선별하는 단계.(d) When synaptic function is improved compared to before treatment with a candidate substance, selecting as a candidate substance for preventing or treating neurological diseases.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those having ordinary knowledge in the technical field to which the present invention belongs from the following description. There will be.
상기와 같은 목적을 달성하기 위해, 본 발명은 하기 단계를 포함하는, 시냅스 기능 장애 평가 방법을 제공한다:In order to achieve the above object, the present invention provides a method for evaluating synaptic dysfunction, comprising the following steps:
(a) 고정 용액으로 동물의 뇌 조직 시료를 고정시키는 단계;(a) immobilizing the brain tissue sample of the animal with a fixation solution;
(b) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(b) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
(c) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (c) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
(d) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(d) fixing the washed sample with a mounting solution; And
(e) 상기 (a) 내지 (d) 단계를 거친 뇌 조직 시료에서 뉴런의 스파인(spine) 길이 및 분포를 분석하는 단계.(e) analyzing the spine length and distribution of neurons in a brain tissue sample subjected to steps (a) to (d).
또한, 본 발명은 하기 단계를 포함하는, 신경계 질환 예방 또는 치료용 후보물질의 스크리닝 방법을 제공한다:In addition, the present invention provides a method of screening a candidate substance for preventing or treating a nervous system disease, comprising the following steps:
(a) 신경계 질환 동물모델에 후보물질을 처리하는 단계;(a) processing the candidate material in an animal model of a nervous system disease;
(b) 상기 동물의 뇌 조직을 투명화 시키는 단계;(b) making the brain tissue of the animal transparent;
(c) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 뇌 조직에서 뉴런의 스파인(spine) 길이 및 분포를 분석하여 시냅스 기능을 평가하는 단계; 및(c) analyzing synaptic function by analyzing the spine length and distribution of neurons in the cleared brain tissue through the Imaris program; And
(d) 시냅스 기능이 후보물질을 처리하기 전에 비해 개선된 경우 신경계 질환 예방 또는 치료용 후보물질로 선별하는 단계.(d) When synaptic function is improved compared to before treatment with a candidate substance, selecting as a candidate substance for preventing or treating neurological diseases.
본 발명의 일 구현예로서, 상기 고정 용액은 수크로오스(sucrose)를 포함할 수 있다.As an embodiment of the present invention, the fixed solution may include sucrose.
본 발명의 다른 구현예로서, 상기 수크로오스(sucrose)는 농도가 20%(w/v) 내지 100%(w/v)일 수 있다.As another embodiment of the present invention, the sucrose may have a concentration of 20% (w/v) to 100% (w/v).
본 발명의 또 다른 구현예로서, 상기 조직 클리어링 용액은 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution), CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.As another embodiment of the present invention, the tissue clearing solution is N-Lauroylsarcosine sodium salt solution, CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) , Urea (urea), and sodium chloride (NaCl).
본 발명의 또 다른 구현예로서, 상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 농도가 1%(v/v) 내지 30%(v/v)이고, CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 10%(w/v) 내지 40%(w/v)이고, 상기 우레아(urea)는 농도가 30%(w/v) 내지 70%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 1%(w/v)일 수 있다.In another embodiment of the present invention, the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v), and CHAPS ( 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/v), and the urea has a concentration. 30% (w/v) to 70% (w/v), and the sodium chloride (NaCl) concentration may be 0.001% (w/v) to 1% (w/v).
본 발명의 또 다른 구현예로서, 상기 세척 용액은 인산완충식염수(phosphate buffer saline; PBS) 및 아지드화나트륨(sodium azide)을 포함할 수 있다.As another embodiment of the present invention, the washing solution may include phosphate buffer saline (PBS) and sodium azide.
본 발명의 또 다른 구현예로서, 상기 아지드화나트륨(sodium azide)은 농도가 0.001%(w/v) 내지 0.5%(w/v)일 수 있다.As another embodiment of the present invention, the sodium azide (sodium azide) may have a concentration of 0.001% (w/v) to 0.5% (w/v).
본 발명의 또 다른 구현예로서, 상기 마운팅 용액은 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.As another embodiment of the present invention, the mounting solution is composed of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include one or more selected from the group.
본 발명의 또 다른 구현예로서, 상기 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 20%(w/v) 내지 60%(w/v)이고, 상기 우레아(urea)는 농도가 10%(w/v) 내지 50%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 3%일 수 있다.As another embodiment of the present invention, the concentration of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/ v), the concentration of urea (urea) is 10% (w/v) to 50% (w/v), and the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3%. .
본 발명의 또 다른 구현예로서, 상기 (e) 단계는 이마리스(Imaris) 프로그램을 이용하는 것일 수 있다.As another embodiment of the present invention, step (e) may be using an Imaris program.
본 발명의 또 다른 구현예로서, 상기 동물의 뇌 조직을 투명화 시키는 단계는 하기 단계를 포함할 수 있다:As another embodiment of the present invention, the step of clearing the brain tissue of the animal may include the following steps:
(가) 고정 용액으로 동물의 뇌 조직 시료를 고정시키는 단계;(A) immobilizing a sample of the brain tissue of the animal with a fixed solution;
(나) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(B) a clearing step of minimizing denaturation of the fluorescent substance in the fixed sample with a tissue clearing solution;
(다) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; 및(C) a washing step of washing the organic matter attached to the sample with minimal degeneration of the fluorescent material with a washing solution; And
(라) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계.(D) fixing the washed sample with a mounting solution.
본 발명에 따른 시냅스 기능 장애 평가 방법은 뇌 조직을 투명화 시키는 단계를 포함하며, 상기 단계에서 뇌 조직을 투명화 시킴으로써 뉴런의 선명한 3차원 이미지를 획득할 수 있고, 이마리스(Imaris) 프로그램을 통해 뉴런의 스파인(spine) 길이 및 분포를 효과적으로 분석하고 정량화하여 시냅스 기능 장애를 평가할 수 있다. 또한, 본 발명의 시냅스 기능 장애 평가 방법은 신경계 질환 동물모델에서 약물의 처리에 따른 뉴런의 스파인 길이 및 분포를 분석함으로써, 신경계 질환 예방 또는 치료용 약물을 스크리닝 하는 데 유용하게 이용될 수 있을 것으로 기대된다.The method for evaluating synaptic dysfunction according to the present invention includes the step of making the brain tissue transparent, and by clearing the brain tissue in the above step, a clear three-dimensional image of the neuron can be obtained, and the neurons of the neurons through the Imaris program. Synaptic dysfunction can be assessed by effectively analyzing and quantifying spine length and distribution. In addition, the method for evaluating synaptic dysfunction of the present invention is expected to be useful for screening drugs for preventing or treating neurological diseases by analyzing the spine length and distribution of neurons according to the treatment of drugs in animal models of neurological diseases. do.
도 1은 본 발명의 일구현예에 따른 투명화 용액을 이용하여 뇌 조직을 투명화하고, 면역 염색하여 획득한 뉴런의 3차원 형광 이미지를 나타낸 도면이다.1 is a view showing a three-dimensional fluorescence image of neurons obtained by clearing and immunostaining brain tissue using a clearing solution according to one embodiment of the present invention.
도 2는 본 발명의 일구현예에 따른 투명화 방법으로 뇌 조직을 투명화 하였을 경우 획득한 뉴런의 3차원 형광 이미지를 CLARITY 방법으로 뇌 조직을 투명화 하였을 경우 획득한 이미지와 비교하여 나타낸 도면이다. FIG. 2 is a diagram comparing a 3D fluorescent image of neurons obtained when the brain tissue is transparent with the transparent method according to an embodiment of the present invention, compared with the image obtained when the brain tissue is transparent with the CLARITY method.
도 3은 본 발명의 일구현예에 따른 투명화 용액을 이용하여 뇌 조직을 투명화하고, 이마리스(Imaris) 프로그램을 통해 뉴런의 스파인(spine) 길이 및 분포를 관찰한 결과를 나타낸 도면이다.3 is a view showing the results of observing the spine length and distribution of neurons through the Imaris program, and transparent the brain tissue using a clearing solution according to an embodiment of the present invention.
본 발명은 하기 단계를 포함하는, 시냅스 기능 장애 평가 방법을 제공한다:The present invention provides a method for evaluating synaptic dysfunction, comprising the following steps:
(a) 고정 용액으로 동물의 뇌 조직 시료를 고정시키는 단계;(a) immobilizing the brain tissue sample of the animal with a fixation solution;
(b) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(b) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
(c) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (c) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
(d) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(d) fixing the washed sample with a mounting solution; And
(e) 상기 (a) 내지 (d) 단계를 거친 뇌 조직 시료에서 뉴런의 스파인(spine) 길이 및 분포를 분석하는 단계.(e) analyzing the spine length and distribution of neurons in a brain tissue sample subjected to steps (a) to (d).
또한, 본 발명은 하기 단계를 포함하는, 신경계 질환 예방 또는 치료용 후보물질의 스크리닝 방법을 제공한다:In addition, the present invention provides a method of screening a candidate substance for preventing or treating a nervous system disease, comprising the following steps:
(a) 신경계 질환 동물모델에 후보물질을 처리하는 단계;(a) processing the candidate material in an animal model of a nervous system disease;
(b) 상기 동물의 뇌 조직을 투명화 시키는 단계;(b) making the brain tissue of the animal transparent;
(c) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 뇌 조직에서 뉴런의 스파인(spine) 길이 및 분포를 분석하여 시냅스 기능을 평가하는 단계; 및(c) analyzing synaptic function by analyzing the spine length and distribution of neurons in the cleared brain tissue through the Imaris program; And
(d) 시냅스 기능이 후보물질을 처리하기 전에 비해 개선된 경우 신경계 질환 예방 또는 치료용 후보물질로 선별하는 단계.(d) When synaptic function is improved compared to before treatment with a candidate substance, selecting as a candidate substance for preventing or treating neurological diseases.
본 발명에서 사용되는 용어 "뉴런(neuron)"은 신경계의 단위로서 신경세포라고도 하고 전기적으로 흥분시킬 수 있으며, 정보를 화학적 혹은 전기적 신호로 바꾸어 전달한다. 뉴런 사이의 신호 전달은 시냅스(synapse)라고 부르는 특화된 연결을 통하여 일어나며, 여러 뉴런이 서로 연결되어 신경망(neural network)을 형성한다. 또한, 뉴런은 크고 동그란 핵을 비롯하여 세포의 기능을 유지하기 위해 필요한 여러 세포소기관(organelle)을 포함하고 있는 신경세포체(cell body, soma), 신경세포체에서 뻗어 나온 것으로 다른 신경세포로부터 정보를 받는 역할을 하는 수상 돌기(dendrite), 및 세포질이 길쭉하게 뻗어 나온 것으로 뉴런이 활동전압이라고 불리는 전기적 신호를 전달하는데 이용하는 축삭(axon)으로 이루어져 있다. The term "neuron" as used in the present invention is a unit of the nervous system, also called a neuron, and can be electrically excited, and converts information into a chemical or electrical signal and transmits it. Signal transmission between neurons occurs through specialized connections called synapses, and several neurons are connected to each other to form a neural network. In addition, neurons extend from neuronal cell bodies (cell bodies, soma), which contain large, round nuclei, and various organelles necessary to maintain the function of cells, and receive information from other neurons. It consists of a dendrite, and an axon used by neurons to transmit an electrical signal called an active voltage.
본 발명에서 사용되는 용어 "스파인(spine)"은 "수상돌기 가시(dendritic spine)"와 동일한 의미로 사용되며, 상기 수상돌기 가시는 신경세포 수상돌기 표면에 존재하는 0.5-2 μm 길이의 작은 돌출 구조로써, 흥분성 시냅스의 시냅스후로 기능한다. 신경세포에 존재하는 수상돌기 가시의 개수 및 크기와 형태는 매우 다양하며, 신경세포의 발달과정 및 개별 시냅스의 활성 정도에 따라 변화한다. 수상돌기 가시 표면에는 시냅스후의 기능에 필요한 다양한 신경전달물질 수용체(Neurotransmitter receptor)가 위치하며, 내부에는 분자신호전달 및 수상돌기 가시 구조 조절 필요한 수백 종류의 다양한 단백질들이 존재한다.The term "spine" used in the present invention is used in the same sense as "dendritic spine", and the dendritic spine is a small protruding structure of 0.5-2 μm length present on the surface of the neuronal dendrite. As, it functions as a post-synaptic synapse. The number, size, and shape of dendritic spines present in neurons vary widely and vary depending on the development process of neurons and the degree of activity of individual synapses. Various neurotransmitter receptors required for postsynaptic function are located on the dendritic spine surface, and there are hundreds of different types of proteins required for molecular signaling and control of the dendritic spine structure.
본 발명에서 사용되는 용어 "시냅스"는 상기 뉴런이 전기적 또는 화학적 신호를 또 다른 세포로 통과시키는 뉴런의 영역을 나타낸다. 시냅스에서, 신호-통과 뉴런(시냅스 전부 뉴런)의 원형질막은 표적(시냅스 후부) 세포의 막과 거의 동격의 상태가 된다.The term "synapse" as used herein refers to a region of a neuron through which the neuron passes electrical or chemical signals to another cell. At synapses, the plasma membrane of signal-passing neurons (all synaptic neurons) is almost identical to the membrane of the target (post-synaptic) cell.
본 발명에 있어서, 상기 후보물질은 신경계 질환 동물모델의 뇌 조직에서 상기 뉴런의 스파인(spine) 길이 및 분포를 분석함으로써 신경계 질환 예방 또는 치료 효능을 확인하기 위한 스크리닝에 이용되는 미지의 물질을 의미하며, 예컨대 화학물질, 단백질, (폴리)뉴클레오타이드, 안티센스-RNA, siRNA (small interference RNA), 또는 천연물 추출물 등이 포함될 수 있으나, 이에 제한되지 않는다. 본 발명에 있어서, 상기 후보물질은 신경계 질환 치료제일 수 있다.In the present invention, the candidate substance refers to an unknown substance used for screening to confirm the efficacy of preventing or treating a nervous system disease by analyzing the spine length and distribution of the neurons in brain tissue of an animal model of a nervous system disease, For example, a chemical, protein, (poly) nucleotide, antisense-RNA, siRNA (small interference RNA), or natural extracts may be included, but are not limited thereto. In the present invention, the candidate material may be a therapeutic agent for a nervous system disease.
본 발명에서 사용되는 용어 “신경계 질환”은 신경세포, 예컨대 뇌세포의 사멸 또는 퇴화가 일시적 또는 오랜 기간에 걸쳐 진행됨으로써 신경 기능의 손실이 일어나고 이에 의해 인지기능, 감각기능, 운동기능, 전신기능의 저하가 일어나는 상태 또는 증상을 의미하며, 뇌졸중(stroke), 치매(dementia), 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 헌팅턴병(Huntington's disease), 피크병(Pick disease), 크로이펠츠-야콥병(Creutzfeld-Jacob disease), 전두측두치매(frontotemporal dementia), 루이치매(dementia with Lewy bodies), 근육위축가쪽경화증(amyotrophic lateral sclerosis; 루게릭병), 피질기저퇴행증(corticobasal degeneration), 다계통위축병(multiple system atrophy), 진행성핵상마비(progressive supranuclearpalsy), 신경계 자가면역질환(neurological autoimmunedisease), 다발성 경화증(multiple sclerosis), 염증성 및 신경병증성 통증(inflammatory and neuropathic pain) 및 뇌혈관질환(neuro vascular disease)으로 이루어진 군으로부터 선택된 어느 하나 이상일 수 있으나, 상기 신경계 질환의 종류에 제한은 없다.The term “nervous system disease” used in the present invention causes loss of nerve function by the death or degeneration of nerve cells, such as brain cells, temporarily or over a long period of time, thereby causing cognitive, sensory, motor, and systemic functions. Refers to the condition or symptom of deterioration, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick disease, Krofelz- Creutzfeld-Jacob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, cortical basal degeneration, multiple system atrophy Multiple system atrophy, progressive supranuclearpalsy, neurological autoimmunedisease, multiple sclerosis, inflammatory and neuropathic pain and neuro vascular disease disease) may be any one or more selected from the group consisting of, but is not limited to the type of the nervous system disease.
본 발명에 있어서, 상기 동물의 뇌 조직을 투명화 시키는 단계는 하기 단계를 포함할 수 있다:In the present invention, the step of clearing the brain tissue of the animal may include the following steps:
(가) 고정 용액으로 동물의 뇌 조직 시료를 고정시키는 단계;(A) immobilizing a sample of the brain tissue of the animal with a fixed solution;
(나) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(B) a clearing step of minimizing denaturation of the fluorescent substance in the fixed sample with a tissue clearing solution;
(다) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; 및(C) a washing step of washing the organic matter attached to the sample with minimal degeneration of the fluorescent material with a washing solution; And
(라) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계.(D) fixing the washed sample with a mounting solution.
본 발명에 있어서, 상기 동물은 그 종류에 특별히 제한이 없으며, 예컨대 인간, 비-인간 영장류, 마우스, 개, 고양이, 토끼, 말, 또는 소일 수 있다. 본 발명의 구체적인 실시예에 따르면 상기 동물은 마우스일 수 있고, Thy-1을 과발현시킨 Thy-1 transgenic 마우스를 이용하여 마우스의 뇌 조직 이미지를 더 잘 관찰할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the animal is not particularly limited in its kind, and may be, for example, a human, non-human primate, mouse, dog, cat, rabbit, horse, or cow. According to a specific embodiment of the present invention, the animal may be a mouse, and a brain tissue image of the mouse may be better observed using a Thy-1 transgenic mouse over-expressing Thy-1, but is not limited thereto.
본 발명에 있어서, 상기 고정 용액은 수크로오스(sucrose)를 포함할 수 있으며, 상기 수크로오스(sucrose)는 농도가 20%(w/v) 내지 100%(w/v), 20%(w/v) 내지 70%(w/v), 20%(w/v) 내지 30%(w/v), 30%(w/v) 내지 40%(w/v), 40%(w/v) 내지 50%(w/v), 50%(w/v) 내지 60%(w/v), 60%(w/v) 내지 70%(w/v), 70%(w/v) 내지 80%(w/v), 또는 80%(w/v) 내지 100%(w/v)일 수 있다. 본 발명의 구체적인 실시예에 따르면 수크로오스의 농도를 20%(w/v) 이상으로 하여 시료를 탈수시키고 PFA(paraformaldehyde)로 유기물간 공유결합된 시료를 좀 더 강하게 고정할 수 있으나, 상기 농도에 한정되는 것은 아니다.In the present invention, the fixed solution may include sucrose (sucrose), the sucrose (sucrose) has a concentration of 20% (w/v) to 100% (w/v), 20% (w/v) To 70% (w/v), 20% (w/v) to 30% (w/v), 30% (w/v) to 40% (w/v), 40% (w/v) to 50 %(w/v), 50%(w/v) to 60%(w/v), 60%(w/v) to 70%(w/v), 70%(w/v) to 80%( w/v), or 80% (w/v) to 100% (w/v). According to a specific embodiment of the present invention, the sample may be dehydrated by setting the concentration of sucrose to 20% (w/v) or more, and the sample covalently bound between organic substances with PFA (paraformaldehyde) may be more strongly fixed, but limited to the concentration. It does not work.
본 발명에 있어서, 상기 조직 클리어링 용액은 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution), CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.In the present invention, the tissue clearing solution is N-Lauroyl sarcosine sodium salt solution (N-Lauroylsarcosine sodium salt solution), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane Sulfonate), urea (urea), and sodium chloride (NaCl).
이 때, 상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 농도가 1%(v/v) 내지 30%(v/v) 또는 3%(v/v) 내지 20%(v/v)일 수 있고, CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 10%(w/v) 내지 40%(w/v) 또는 10%(w/v) 내지 30%(w/v)일 수 있고, 상기 우레아(urea)는 농도가 30%(w/v) 내지 70%(w/v) 또는 40%(w/v) 내지 60%(w/v)일 수 있고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 1%(w/v) 또는 0.01%(w/v) 내지 1%(w/v)일 수 있으며, 본 발명의 구체적인 실시예에 따르면 상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 4%(v/v) 또는 15%(v/v), CHAPS는 20%(w/v), 우레아는 50%(w/v)일 수 있고, 염화나트륨은 농도를 0.1%(w/v) 내지 0.5%(w/v)로 하여 삼투압에 의한 조직의 변형과 ion strength를 안정화시켜 시료에 있는 형광 물질의 변성을 최소화 할 수 있으나, 상기 농도에 제한되는 것은 아니다.In this case, the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v) or 3% (v/v) to 20. % (v/v), and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/ v) or 10% (w/v) to 30% (w/v), wherein the urea has a concentration of 30% (w/v) to 70% (w/v) or 40% (w) /v) to 60% (w/v), and the concentration of sodium chloride (NaCl) is 0.001% (w/v) to 1% (w/v) or 0.01% (w/v) to 1% ( w/v), and according to a specific embodiment of the present invention, the N-Lauroylsarcosine sodium salt solution is 4% (v/v) or 15% (v/v) ), CHAPS may be 20% (w/v), urea may be 50% (w/v), and sodium chloride has a concentration of 0.1% (w/v) to 0.5% (w/v), resulting in osmotic pressure. Stabilization and ion strength can be stabilized to minimize denaturation of the fluorescent material in the sample, but is not limited to the concentration.
본 발명에 있어서, 상기 (b) 조직 클리어링 용액을 이용하는 투명화 단계는 4%(v/v)의 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)을 포함한 조직 클리어링 용액을 이용하는 제 1 투명화 단계, 또는 15%(v/v) 의 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)을 포함한 조직 클리어링 용액을 이용하는 제 2 투명화 단계를 포함할 수 있으며, 본 발명의 구체적인 실시예에 따르면, 상기 제 1 투명화 단계 이후 세척 단계, 제 2 투명화 단계, 및 세척 단계를 순서대로 실시할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the step (b) of the clearing using the tissue clearing solution uses a tissue clearing solution containing 4% (v/v) of N-Lauroylsarcosine sodium salt solution. The first clearing step, or may include a second clearing step using a tissue clearing solution comprising 15% (v/v) of N-Lauroylsarcosine sodium salt solution. According to a specific embodiment of the invention, after the first transparent step, the washing step, the second transparent step, and the washing step may be performed in order, but is not limited thereto.
본 발명에 있어서, 상기 세척 용액은 인산완충식염수(phosphate buffer saline; PBS) 및 아지드화나트륨(sodium azide)을 포함할 수 있으며, 상기 아지드화나트륨(sodium azide)은 농도가 0.001%(w/v) 내지 0.5%(w/v) 또는 0.01%(w/v) 내지 0.5%(w/v)일 수 있고, 본 발명의 구체적인 실시예에 따르면, 상기 아지드화나트륨(sodium azide)은 농도를 0.1%(w/v)로 하여 투명화 후 탈수된 생체조직 시료에 최대 30%까지 함수율을 증가시킨 후 15%를 탈수하여 조직에 붙어있는 이미징에 방해되는 유기물을 세척할 수 있으나, 상기 농도에 제한되지는 않는다.In the present invention, the washing solution may include phosphate buffered saline (PBS) and sodium azide, and the sodium azide has a concentration of 0.001% (w /v) to 0.5% (w/v) or 0.01% (w/v) to 0.5% (w/v), according to a specific embodiment of the present invention, the sodium azide (sodium azide) is After clearing the concentration to 0.1% (w/v), after increasing the water content up to 30% in a dehydrated biotissue sample, dehydrating 15% to wash the organic matter that interferes with imaging attached to the tissue, but the concentration It is not limited to.
본 발명에 있어서, 상기 마운팅 용액은 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.In the present invention, the mounting solution is one selected from the group consisting of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include the above.
이 때, 상기 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 20%(w/v) 내지 60%(w/v) 또는 30%(w/v) 내지 50%(w/v)일 수 있고, 상기 우레아(urea)는 농도가 10%(w/v) 내지 50%(w/v) 또는 20%(w/v) 내지 50%(w/v)일 수 있고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 3% 또는 0.01%(w/v) 내지 2%일 수 있으며, 본 발명의 구체적인 실시예에 따르면 상기 마운팅 용액의 굴절률을 1.45로 맞추기 위해 상기 CHAPS 및 우레아의 조성은 각각 40%(w/v), 40%(w/v)로 포함할 수 있으며, 염화나트륨(NaCl)의 농도를 0.1 내지 1%(w/v)로 포함할 수 있으나, 상기 농도에 제한되는 것은 아니다.At this time, the concentration of the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/v) or 30% ( w/v) to 50% (w/v), and the urea has a concentration of 10% (w/v) to 50% (w/v) or 20% (w/v) to 50%. (w/v), the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3% or 0.01% (w/v) to 2%, according to a specific embodiment of the present invention In order to set the refractive index of the mounting solution to 1.45, the composition of the CHAPS and urea may include 40% (w/v) and 40% (w/v), respectively, and the concentration of sodium chloride (NaCl) is 0.1 to 1%. (w/v), but is not limited to the concentration.
본 발명에 있어서, 상기 (e) 단계는 이마리스(Imaris) 프로그램을 이용하는 것일 수 있다.In the present invention, step (e) may be using an Imaris program.
본 발명의 일 실시예에서는, Thy-1 transgenic 마우스의 뇌 조직을 투명화하기 위한 용액을 제조하고, 상기 용액을 사용하여 Thy-1 transgenic 마우스의 뇌 조직을 투명화한 후 면역 염색하여 뉴런의 3차원 면역 염색 이미지를 획득하였다(실시예 1 참조).In one embodiment of the present invention, a solution for clearing the brain tissue of a Thy-1 transgenic mouse is prepared, and the brain tissue of the Thy-1 transgenic mouse is cleared using the solution, followed by immunostaining, to provide three-dimensional immunity of neurons. Dyed images were obtained (see Example 1).
본 발명의 다른 실시예에서는, 이마리스(Imaris) 프로그램을 이용하여 상기 투명화한 뇌 조직에서 뉴런의 스파인(spine) 길이 및 분포를 관찰하였으며, 이를 통해 시냅스의 기능 장애 여부를 확인할 수 있을 것으로 예상되었다(실시예 2 참조).In another embodiment of the present invention, the spine length and distribution of neurons were observed in the transparent brain tissue using the Imaris program, and it was expected to be able to confirm whether or not synaptic dysfunction occurred. (See Example 2).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예Example 1. Thy-1 1. Thy-1 transgenictransgenic 마우스의 뇌 조직 투명화 및 뉴런의 3차원 면역 염색 이미지 획득 Transparent brain tissue and 3D immunostaining images of neurons
1-1. 고정 용액, 조직 1-1. Fixed solution, tissue 클리어링Clearing 용액, 세척 용액, 및 Solution, washing solution, and 마운팅Mounting 용액의 제조 Preparation of solution
뇌 조직을 투명화시키기 위해 먼저 조직 투명화를 위해 필요한 고정 용액, 조직 클리어링 용액, 세척 용액, 및 마운팅 용액을 제조하였으며, 상기 용액의 구성 성분은 하기 표 1에 나타내었다.To clear the brain tissue, first, a fixation solution, a tissue clearing solution, a washing solution, and a mounting solution required for tissue clearing were prepared, and the components of the solution are shown in Table 1 below.
구체적으로, 생체조직 시료를 탈수시키고 PFA(paraformaldehyde)로 유기물간 공유결합된 시료를 좀 더 강하게 고정시키기 위해, 농도가 20%(w/v) 이상인 수크로오스(sucrose)를 구성성분으로 하는 고정 용액(fixing solution)을 제조하였다. Specifically, in order to dehydrate the biological tissue sample and to more strongly fix the sample covalently bound between organic materials with PFA (paraformaldehyde), a fixed solution containing sucrose having a concentration of 20% (w/v) or more as a component ( fixing solution).
또한, 삼투압에 의한 조직의 변형과 ion strength를 안정화시켜 생체조직 시료에 있는 형광 물질의 변성을 최소화 하기 위해, 4%(v/v) 또는 15%(v/v) 농도의 N-Lauroylsarcosine sodium salt solution, 20%(w/v) CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트) 및 50%(w/v) 우레아(urea)에 0.1 내지 0.5%(w/v) 농도의 염화나트륨(NaCl)을 추가하여, 본 발명의 조직 클리어링 용액(tissue clearing solution)을 제조하였다. In addition, N-Lauroylsarcosine sodium salt at a concentration of 4% (v/v) or 15% (v/v) to minimize the degeneration of the fluorescent substance in the biological tissue sample by stabilizing the tissue deformation and ion strength by osmotic pressure. solution, 0.1% to 0.5% in 20% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 50% (w/v) urea By adding (w/v) concentration of sodium chloride (NaCl), a tissue clearing solution of the present invention was prepared.
이어서, 투명화 후 탈수된 생체조직 시료에 최대 30%까지 함수율을 증가시킨 후 15%를 탈수하여 조직에 붙어있는 이미징에 방해되는 유기물을 세척시킬 수 있도록 인산완충식염수(phosphate buffer saline; PBS)에 0.1%(w/v)의 아지드화나트륨(sodium azide)을 첨가하여 세척 용액(washing solution)을 제조하였다. Subsequently, 0.1% in phosphate buffer saline (PBS) to increase the water content to a maximum of 30% in dehydrated biotissue samples after clarification and then dehydrate 15% to wash organic matters that interfere with imaging attached to the tissues. A washing solution was prepared by adding% (w/v) sodium azide.
또한, 40%(w/v)의 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 40%(w/v) 우레아(urea), 및 0.1 내지 1%(w/v) NaCl로 마운팅 용액(mounting solution)을 제조하여 굴절률을 1.45로 맞추었다.In addition, 40% (w/v) of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 40% (w/v) urea, and 0.1 to A mounting solution was prepared with 1% (w/v) NaCl to adjust the refractive index to 1.45.
구성Configuration 구성 성분Composition
1One Fixing solutionFixing solution Sucrose(20%(w/v)이상)Sucrose (more than 20% (w/v))
22 Tissue clearing solution Tissue clearing solution N-Lauroylsarcosine sodium salt solution(4%(v/v) 또는 15%(v/v)), CHAPS(20%(w/v)), Urea(50%(w/v)), NaCl(0.1 내지 0.5%(w/v))N-Lauroylsarcosine sodium salt solution (4% (v/v) or 15% (v/v)), CHAPS (20% (w/v)), Urea (50% (w/v)), NaCl (0.1 to 0.5% (w/v))
33 Washing solutionWashing solution PBS, sodium azied(0.1%(w/v))PBS, sodium azied (0.1% (w/v))
44 Mounting solutionMounting solution CHAPS(40%(w/v)), Urea(40%(w/v)), NaCl(0.1 내지 1%(w/v))CHAPS (40% (w/v)), Urea (40% (w/v)), NaCl (0.1 to 1% (w/v))
1-2. 뇌 조직 투명화 및 뉴런의 3차원 면역 염색 이미지 획득1-2. Transparent tissue and acquisition of 3D immunostaining images of neurons
뇌 조직의 뉴런 3차원 면역 염색 이미지를 확인하기 위해 상기 실시예 1-1에서 제조한 용액(표 1 참조)을 사용하여 Thy-1 GFP transgenic 마우스의 뇌 조직 시료를 투명화 하였다. To confirm the three-dimensional immunostaining image of neurons in brain tissue, a brain tissue sample of Thy-1 GFP transgenic mouse was clarified using the solution prepared in Example 1-1 (see Table 1).
구체적으로, The Jackson Laboratory에서 Thy-1 GFP transgenic 마우스를 구입하였으며, 상기 마우스의 brain 시료를 얻기 위해 마취가스로 이소플루란 (isoflurane)을 이용하여 흡입 마취하고, 100% 산소와 혼합하였다. 또한, 마취 후 개복하여 30 ml PBS(pH 7.2)로 3-5분 동안 cardiac perfusion 하고, 다시 30-50 ml의 4% paraformaldehyde로 20-30분 동안 perfusion 하여 마우스의 뇌 조직 시료를 얻었다. Specifically, a Thy-1 GFP transgenic mouse was purchased from The Jackson Laboratory, and inhaled anesthesia using isoflurane as an anesthetic gas to obtain a brain sample of the mouse, and mixed with 100% oxygen. In addition, after anesthesia, the cells were opened and cardiac perfusion was performed with 30 ml PBS (pH 7.2) for 3-5 minutes, followed by perfusion with 30-50 ml of 4% paraformaldehyde for 20-30 minutes to obtain brain tissue samples of mice.
그리고 나서, 상기로부터 수득한 마우스의 뇌 조직 시료를 고정 용액(fixing solution)에 넣고 4℃/50 rpm에서 침전될 때까지 shaking incubation 하고, 침전된 뇌 조직 시료를 4%(v/v) N-Lauroylsarcosine sodium salt solution을 포함하는 조직 클리어링 용액(tissue clearing solution)에 넣고 35℃/50 rpm으로 36시간 동안 shaking incubation하고, 동일한 조건으로 1회 반복하였다.Then, the brain tissue sample of the mouse obtained from the above was placed in a fixing solution, shaking incubation until precipitation at 4°C/50 rpm, and the precipitated brain tissue sample was 4% (v/v) N- It was added to a tissue clearing solution containing lauroylsarcosine sodium salt solution, shaking incubation for 36 hours at 35°C/50 rpm, and repeated once under the same conditions.
상기 반응이 끝난 뇌 조직 시료에 50ml의 세척 용액(washing solution)을 넣고 4℃/50 rpm에서 4시간 동안 shaking incubation한 후(3회 반복), 15%(v/v)의 N-Lauroylsarcosine sodium salt solution을 포함하는 조직 클리어링 용액(tissue clearing solution)에 넣고 상기와 동일하게 shaking incubation 하고, 이를 세척 용액(washing solution)으로 다시 washing하였다. After 50 ml of washing solution was added to the brain tissue sample after the reaction, shaking incubation was performed at 4°C/50 rpm for 4 hours (repeat 3 times), and 15% (v/v) of N-Lauroylsarcosine sodium salt It was placed in a tissue clearing solution containing a solution, shaking incubation as described above, and washed again with a washing solution.
상기 washing이 끝난 뇌 조직 시료는 마운팅 용액(mounting solution)에 넣고 35℃/50 rpm으로 12시간 shaking incubation 하고, 동일한 조건으로 1회 반복하였다.The washed brain tissue sample was placed in a mounting solution, shaking incubation at 35°C/50 rpm for 12 hours, and repeated once under the same conditions.
그런 다음, 상기 마운팅 용액으로 처리한 뇌 조직 시료를 1200 rpm에서 30분 동안 centrifuge하여 시료 내 bubble을 제거하고, 뇌 조직 시료를 image chamber에서 보관하거나 1X PBS에 4℃에서 보관하였다.Then, the brain tissue sample treated with the mounting solution was centrifuge for 30 minutes at 1200 rpm to remove bubbles in the sample, and the brain tissue sample was stored in an image chamber or stored at 4°C in 1X PBS.
상기 방법으로 투명화가 완료된 뇌 조직의 Thy-1 GFP의 형광량을 lightsheet 현미경을 이용하여 측정하였다. 그 결과, 도 1에 나타낸 바와 같이 상기 방법으로 투명화한 뇌 조직에서 선명한 뉴런의 3차원 형광 이미지를 확인할 수 있었다.The fluorescence amount of Thy-1 GFP of the brain tissue, which had been cleared by the above method, was measured using a lightsheet microscope. As a result, as shown in FIG. 1, it was possible to confirm a three-dimensional fluorescence image of clear neurons in the brain tissue transparent by the above method.
또한, 상기 방법으로 투명화한 뇌 조직과 종래 조직 투명화 방법인 CLARITY 방법으로 투명화한 뇌 조직에서의 뉴런 3차원 면역 염색 이미지를 비교하였으며, 그 결과, 도 2에 나타낸 바와 같이 기존의 조직 투명화 방법인 CLARITY 방법으로 뇌 조직을 투명화 시켰을 때(도 2의 좌측 이미지)에 비해, 표 1에 나타낸 본 발명의 조직 투명화 용액을 이용하여 뇌 조직을 투명화 시켰을 때(도 2의 우측 이미지), 더 선명한 뉴런의 3차원 형광 이미지를 얻을 수 있었다.In addition, the three-dimensional immunostaining images of neurons in the brain tissues transparent with the above method and the brain tissues transparent with the conventional tissue transparency method CLARITY were compared, and as a result, as shown in FIG. 2, the conventional tissue transparent method CLARITY. When the brain tissue was cleared using the tissue clearing solution of the present invention shown in Table 1 compared to when the brain tissue was cleared by the method (left image in FIG. 2), 3 of the clearer neurons A dimensional fluorescent image was obtained.
실시예Example 2. 2. 이마리스Imaris (( ImarisImaris ) 프로그램을 통한 뉴런의 스파인 길이 및 분포 분석) Analysis of spine length and distribution of neurons through the program
상기 실시예 1-2의 방법으로 뇌 조직을 투명화 시킨 후 이마리스(Imaris) 3D 프로그램을 사용하여 3차원으로 나타난 뉴런의 형광 이미지에서 뉴런의 spine 길이 및 분포를 관찰하였다.After the brain tissue was made transparent by the method of Example 1-2, the spine length and distribution of neurons were observed in a fluorescence image of neurons in three dimensions using an Imaris 3D program.
구체적으로, 상기 실시예 1-2에서 lightsheet현미경으로 관찰한 3차원 이미지를 Imaris filaments를 이용하여 분석하고자 하는 영역을 설정 후 뉴런의 신경세포체(cell body)와 수상돌기(dendrite)를 구분하였다. 시작점(수상돌기의 지름을 설정)에서 끝나는 지점까지의 선택 영역과 3D 이미지의 매칭을 확인하고, 영상의 형광량을 분석하여 수상돌기의 지름 길이를 정확하게 관찰하였고, 수상돌기의 spine은 도 3에 나타낸 바와 같이 파란색으로 나타나는 것을 확인하였으며, Imaris 프로그램에 내장되어 있는 설정으로 spine의 전체 수, 모양, 분지 등을 분석하였다.Specifically, after setting the region to be analyzed using Imaris filaments in the 3D image observed with the lightsheet microscope in Example 1-2, the neuron cell body and dendrite of the neuron were separated. The selection area from the starting point (set the diameter of the dendrite) to the ending point was checked, the 3D image was matched, and the fluorescence of the image was analyzed to accurately observe the diameter length of the dendrite, and the spine of the dendrite is shown in FIG. 3. As shown, it was confirmed to appear in blue, and the total number, shape, and branching of the spine were analyzed using the settings built into the Imaris program.
수상돌기 spine은 신경세포의 수상돌기에 존재하는 흥분성 신호를 받아들이는 작은 돌출부로서 크기와 모양이 다양하며, 일반적으로 크기가 큰 spine은 그에 비례하는 큰 시냅스 틀을 형성하고 더 다양한 소기관을 가진다. 시냅스후 치밀질(Postsynaptic density)은 보통 spine head에 위치하는 후시냅스 막 근처의 구조물로서 spine 표면적의 10% 정도를 차지하고 있으며, 이러한 spine head의 크기는 시냅스후 치밀질의 면적, 후시냅스 수용체의 수, 시냅스 낭의 수와 비례하기 때문에 spine의 생성 및 성장 기전이 시냅스의 신호전달의 강도와 밀접한 관계가 있다.The dendritic spine is a small protrusion that receives the excitatory signal present in the dendrite of a neuron and has various sizes and shapes. Generally, a large spine forms a large synaptic frame proportional to it and has more diverse organelles. Postsynaptic density is a structure near the postsynaptic membrane, usually located on the spine head, occupying about 10% of the surface area of the spine. Since it is proportional to the number of synaptic sacs, the mechanism of spine formation and growth is closely related to the intensity of synaptic signaling.
따라서, 상기와 같이 수상돌기 및 spine의 길이, 분포 등을 분석함으로써 시냅스의 기능 장애를 평가할 수 있을 것으로 예상되었다.Therefore, it was expected to be able to evaluate synaptic dysfunction by analyzing the length, distribution, and the like of the dendrites and spines as described above.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.
본 발명에 따른 시냅스 기능 장애 평가 방법은 뉴런의 스파인(spine) 길이 및 분포를 효과적으로 분석하고 정량화하여 시냅스 기능 장애를 확인하는데 이용할 수 있으며, 신경계 질환 예방 또는 치료용 약물을 스크리닝 하는 데에도 유용하게 이용할 수 있을 것으로 기대된다.The method for evaluating synaptic dysfunction according to the present invention can be effectively used to identify synaptic dysfunction by effectively analyzing and quantifying the spine length and distribution of neurons, and is also useful for screening drugs for preventing or treating neurological diseases. It is expected to be possible.

Claims (12)

  1. 하기 단계를 포함하는, 시냅스 기능 장애 평가 방법:A method for evaluating synaptic dysfunction comprising the following steps:
    (a) 고정 용액으로 동물의 뇌 조직 시료를 고정시키는 단계;(a) immobilizing the brain tissue sample of the animal with a fixation solution;
    (b) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(b) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
    (c) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (c) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
    (d) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(d) fixing the washed sample with a mounting solution; And
    (e) 상기 (a) 내지 (d) 단계를 거친 뇌 조직 시료에서 뉴런의 스파인(spine) 길이 및 분포를 분석하는 단계.(e) analyzing the spine length and distribution of neurons in a brain tissue sample subjected to steps (a) to (d).
  2. 제1항에 있어서,According to claim 1,
    상기 고정 용액은 수크로오스(sucrose)를 포함하는 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The fixed solution is characterized in that it contains sucrose (sucrose), synaptic dysfunction evaluation method.
  3. 제2항에 있어서,According to claim 2,
    상기 수크로오스(sucrose)는 농도가 20%(w/v) 내지 100%(w/v)인 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The sucrose (sucrose) is characterized in that the concentration is 20% (w / v) to 100% (w / v), synaptic dysfunction evaluation method.
  4. 제1항에 있어서,According to claim 1,
    상기 조직 클리어링 용액은 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution), CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함하는 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The tissue clearing solution is N-Lauroylsarcosine sodium salt solution, CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea (urea), and sodium chloride (NaCl), characterized in that it comprises at least one selected from the group consisting of, synaptic dysfunction evaluation method.
  5. 제4항에 있어서,According to claim 4,
    상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 농도가 1%(v/v) 내지 30%(v/v)이고, CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 10%(w/v) 내지 40%(w/v)이고, 상기 우레아(urea)는 농도가 30%(w/v) 내지 70%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 1%(w/v)인 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v), and CHAPS (3-[(3-Colamido Propyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/v), and the urea has a concentration of 30% (w/v) to 70 % (w / v), the sodium chloride (NaCl) concentration is 0.001% (w / v) to 1% (w / v), characterized in that synaptic dysfunction evaluation method.
  6. 제1항에 있어서,According to claim 1,
    상기 세척 용액은 인산완충식염수(phosphate buffer saline; PBS) 및 아지드화나트륨(sodium azide)을 포함하는 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The washing solution is characterized in that it comprises a phosphate buffer saline (PBS) and sodium azide (sodium azide), synaptic dysfunction evaluation method.
  7. 제6항에 있어서,The method of claim 6,
    상기 아지드화나트륨(sodium azide)은 농도가 0.001%(w/v) 내지 0.5%(w/v)인 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The sodium azide (sodium azide) is characterized in that the concentration is 0.001% (w / v) to 0.5% (w / v), synaptic dysfunction evaluation method.
  8. 제1항에 있어서,According to claim 1,
    상기 마운팅 용액은 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함하는 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The mounting solution comprises one or more selected from the group consisting of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl) Characterized by, synaptic dysfunction evaluation method.
  9. 제8항에 있어서,The method of claim 8,
    상기 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 20%(w/v) 내지 60%(w/v)이고, 상기 우레아(urea)는 농도가 10%(w/v) 내지 50%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 3%인 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 20% (w/v) to 60% (w/v), and the urea Is a concentration of 10% (w / v) to 50% (w / v), the sodium chloride (NaCl) is characterized in that the concentration is 0.001% (w / v) to 3%, synaptic dysfunction evaluation method.
  10. 제1항에 있어서,According to claim 1,
    상기 (e) 단계는 이마리스(Imaris) 프로그램을 이용하는 것을 특징으로 하는, 시냅스 기능 장애 평가 방법.The step (e), characterized in that using the Imaris (Imaris) program, synaptic dysfunction evaluation method.
  11. 하기 단계를 포함하는, 신경계 질환 예방 또는 치료용 후보물질의 스크리닝 방법:A screening method for candidates for preventing or treating neurological diseases, comprising the following steps:
    (a) 신경계 질환 동물모델에 후보물질을 처리하는 단계;(a) processing the candidate material in an animal model of a nervous system disease;
    (b) 상기 동물의 뇌 조직을 투명화 시키는 단계;(b) making the brain tissue of the animal transparent;
    (c) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 뇌 조직에서 뉴런의 스파인(spine) 길이 및 분포를 분석하여 시냅스 기능을 평가하는 단계; 및(c) analyzing synaptic function by analyzing the spine length and distribution of neurons in the cleared brain tissue through the Imaris program; And
    (d) 시냅스 기능이 후보물질을 처리하기 전에 비해 개선된 경우 신경계 질환 예방 또는 치료용 후보물질로 선별하는 단계.(d) When synaptic function is improved compared to before treatment with a candidate substance, selecting as a candidate substance for preventing or treating neurological diseases.
  12. 제11항에 있어서,The method of claim 11,
    상기 동물의 뇌 조직을 투명화 시키는 단계는 하기 단계를 포함하는 것을 특징으로 하는, 스크리닝 방법:The step of making the animal brain tissue transparent comprises the following steps:
    (가) 고정 용액으로 동물의 뇌 조직 시료를 고정시키는 단계;(A) immobilizing a sample of the brain tissue of the animal with a fixed solution;
    (나) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(B) a clearing step of minimizing denaturation of the fluorescent substance in the fixed sample with a tissue clearing solution;
    (다) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; 및(C) a washing step of washing the organic matter attached to the sample with minimal degeneration of the fluorescent material with a washing solution; And
    (라) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계.(D) fixing the washed sample with a mounting solution.
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