WO2020159264A1 - Procédé d'évaluation d'un dysfonctionnement synaptique à l'aide d'une transparisation du tissu cérébral - Google Patents

Procédé d'évaluation d'un dysfonctionnement synaptique à l'aide d'une transparisation du tissu cérébral 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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PCT/KR2020/001442
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Korean (ko)
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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

La présente invention concerne un procédé d'évaluation d'un dysfonctionnement synaptique à l'aide d'une transparisation du tissu cérébral et un procédé d'évaluation d'un dysfonctionnement synaptique, selon la présente invention, comprend une étape consistant à rendre un tissu cérébral transparent, une image tridimensionnelle claire d'un neurone pouvant être obtenue par la transparisation du tissu cérébral et un dysfonctionnement synaptique pouvant être évalué par l'analyse et la quantification efficaces de la longueur et de la distribution des épines du neurone par l'intermédiaire du programme Imaris. De plus, on s'attend à ce que le procédé de la présente invention pour évaluer un dysfonctionnement synaptique soit utile pour prévenir des troubles neurologiques et pour cribler un médicament pour le traitement par analyse de la longueur et de la distribution des épines d'un neurone en fonction du traitement du médicament dans des modèles animaux d'un trouble neurologique.
PCT/KR2020/001442 2019-02-01 2020-01-30 Procédé d'évaluation d'un dysfonctionnement synaptique à l'aide d'une transparisation du tissu cérébral WO2020159264A1 (fr)

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Citations (5)

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JP2015062407A (ja) * 2013-08-30 2015-04-09 公益財団法人東京都医学総合研究所 精神・神経疾患モデル動物
US20170182012A1 (en) * 2013-12-27 2017-06-29 National University Corporation Tokyo Medical And Dental University Method for diagnosis of alzheimer's disease and frontotemporal lobar degeneration, diagnostic agent, therapeutic agent, and screening method for said agents
US20180031452A1 (en) * 2015-03-18 2018-02-01 Riken Method for observing biological material and clearing method
KR101849704B1 (ko) * 2017-08-21 2018-04-18 한국화학연구원 생체 조직의 투명화 전처리 방법 및 이를 포함하는 생체 조직의 투명화 방법
KR20180060494A (ko) * 2016-11-29 2018-06-07 박순현 생체 조직 투명화용 조성물 및 이를 이용한 생체 조직 투명화 방법

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KR20150085578A (ko) 2014-01-16 2015-07-24 서울대학교산학협력단 지방줄기세포 추출물을 포함하는 신경계 질환 치료 또는 예방용 약학 조성물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015062407A (ja) * 2013-08-30 2015-04-09 公益財団法人東京都医学総合研究所 精神・神経疾患モデル動物
US20170182012A1 (en) * 2013-12-27 2017-06-29 National University Corporation Tokyo Medical And Dental University Method for diagnosis of alzheimer's disease and frontotemporal lobar degeneration, diagnostic agent, therapeutic agent, and screening method for said agents
US20180031452A1 (en) * 2015-03-18 2018-02-01 Riken Method for observing biological material and clearing method
KR20180060494A (ko) * 2016-11-29 2018-06-07 박순현 생체 조직 투명화용 조성물 및 이를 이용한 생체 조직 투명화 방법
KR101849704B1 (ko) * 2017-08-21 2018-04-18 한국화학연구원 생체 조직의 투명화 전처리 방법 및 이를 포함하는 생체 조직의 투명화 방법

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