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WO2019039438A1 - Procédé d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain, et dispositif d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain - Google Patents

Procédé d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain, et dispositif d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain Download PDF

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WO2019039438A1
WO2019039438A1 PCT/JP2018/030683 JP2018030683W WO2019039438A1 WO 2019039438 A1 WO2019039438 A1 WO 2019039438A1 JP 2018030683 W JP2018030683 W JP 2018030683W WO 2019039438 A1 WO2019039438 A1 WO 2019039438A1
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Prior art keywords
cerebral infarction
human mammal
brain
observation
carotid artery
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Japanese (ja)
Inventor
矢田　健一郎
秀和 冨本
明 溝口
昌樹 稲垣
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Mie University NUC
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Mie University NUC
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/02—Detecting, measuring or recording for evaluating the cardiovascular system, e.g. pulse, heart rate, blood pressure or blood flow
    • A61B5/026—Measuring blood flow
    • A61B5/0285—Measuring or recording phase velocity of blood waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/417—Imidazole-alkylamines, e.g. histamine, phentolamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a method for observing a non-human mammalian cerebral infarction model and a device for observing a non-human mammalian cerebral infarction model.
  • Cerebral infarction means a condition that causes cerebral ischemia mainly due to occlusion or narrowing of blood vessels that nourish the brain, and causes necrosis of brain tissue due to oxygen and nutrient deficiency.
  • the number of patients with cerebral infarction is about 1.5 million, and it is said that about 500,000 people develop each year. Cerebral infarction is located in the fifth place among Japanese causes of death. When suffering from a cerebral infarction, long-term care is often required, leaving sequelae, even if you escape from acute death. Cerebral infarction accounts for about 30% of the causes of bedridden and has become a disease with major problems in terms of care and welfare.
  • cerebral infarction model By establishing a cerebral infarction model and an observation method that can be comprehensively observed, the pathogenesis of cerebral infarction including elucidation of new pathologies that could not be grasped by conventional observation methods, and more reliable drug efficacy determination, and establishment of treatment methods It is expected that it will be useful to
  • the present invention relates to a transient forebrain ischemia model and a cortical cerebral infarction model of cerebral ischemic lesions by occlusion and reopening of the common carotid artery by remote control, and a brain of the model which was impossible by the conventional method.
  • the present invention provides various observation methods and devices capable of comprehensively capturing minute changes and transient changes therein, and drug screening methods using the model animals and the observation methods and devices.
  • the present inventor observed that the abnormal contraction (spasm) of the cerebral blood vessels occurs with cerebral ischemia in the observation using the above-mentioned model and the abnormal contraction is a major cause of circulatory disorder at the time of cerebral ischemia.
  • a drug for preventing contraction is a drug for treating cerebral infarction. Furthermore, they found that the pathogenesis of abnormal contraction was sympathetic activation via alpha adrenergic receptors and extracellular influx of calcium via calcium channels.
  • the present invention provides abnormal contraction / preventive drugs / therapeutic agents, efficacy screening methods for abnormal contraction / prevention / treatment, and the like.
  • the present inventor provides a carotid artery occlusion / stroke model by remote control of rodents in order to conduct a comprehensive observation.
  • "Overall” means capturing a cerebral infarction lesion temporally and spatially.
  • Temporally means before observation of blood vessel occlusion, during blood vessel occlusion, continuously observing a series of morphological changes up to reperfusion continuously, and spatially means from one cell level to capillary from artery. It means a method of three-dimensionally observing up to the entire image of the vascular system connected to the vein through the blood vessel.
  • the model it is possible to observe the entire process without interruption, from the normal state before the stenosis to the vessel occlusion and the vessel reopening. According to this method, in the conventional research method, it is possible to reliably capture the transient change that occurs immediately after the onset of cerebral infarction or at the time of reopening, which could not be captured.
  • various observation methods and devices for observing the model are provided.
  • Direct observation without interruption of a series of pathologies of cerebral infarction including before the onset of cerebral infarction and during ischemia, using three kinds of methods of two-photon microscope, two-dimensional laser blood flow meter and stereomicroscope Provide a way to In the observation method using a two-photon microscope, cerebral infarction lesions can be observed almost exhaustively (changes including changes in one cell level, vascular system construction, flow of red blood cells and white blood cells in blood vessels, and the like).
  • the observation method using a two-dimensional laser blood flow meter it is possible to simply observe changes in cerebral blood flow throughout the brain from before the onset of infarct to after reopening.
  • the present invention has been made in view of the above-mentioned problems, and an object thereof is to provide an observation method by a non-human mammal cerebral infarction model and an observation apparatus by a non-human mammal cerebral infarction model.
  • the first invention for solving the above problems is a two-photon laser microscope, a two-dimensional laser blood in a bilateral common carotid artery remote occlusion model, a cortical infarction model, of an animal treated so as to enable brain observation of a non-human mammal.
  • the non-human mammal is preferably one selected from the group consisting of a mouse, a rat, a rabbit and a monkey. At this time, it is preferable that the non-human mammal is a GFP non-human mammal that expressed green fluorescent protein (GFP) systemically.
  • GFP green fluorescent protein
  • a method of developing a cerebral infarction drug according to the present invention is characterized by examining the influence of a drug on cerebral infarction using the above-mentioned observation method.
  • the preventive and / or therapeutic agent for cerebral infarction according to the present invention is characterized by being developed by the above-mentioned development method.
  • the preventive and / or therapeutic agent for cerebral infarction is an ⁇ -adrenoceptor antagonist, and is preferably phentolamine mesylate and a related compound thereof.
  • a preventive and / or therapeutic agent for cerebral infarction is a calcium channel blocker, preferably nicardipine and its analogues.
  • a non-human mammal observation apparatus relates to a non-human mammal, a fixation base for fixing a non-human mammal's cranial skull under anesthesia, and blood flow of a carotid artery of the non-human mammal.
  • the non-human mammal is preferably a mouse.
  • the imaging apparatus includes a resonant scanner as a scanner and a cooled high sensitivity gallium arsenide phosphide (GaAsP) detector as a detector.
  • GaAsP gallium arsenide phosphide
  • a comprehensive analysis method of acquired data is an image processing method characterized in that detection of a minute morphological change is facilitated.
  • Visually identifying minor changes in the VOI, including the vast amount of data, is nearly impossible.
  • the two images are displayed in different colors, a superimposed image of the two images is created, and a change site is identified from the color difference.
  • the morphologically unchanged part is a mixture of two colors, and the morphologically changed part is represented by a single color, so that the site of change can be easily identified.
  • alignment two-dimensional or three-dimensional
  • the exhaustive analysis method of volume data is an exhaustive analysis method of volume data captured by the above-mentioned cerebral infarction disease state non-human mammal observation device, and an analysis method comprising the following steps, (1) A data acquisition step of obtaining two types of the volume data different qualitatively; (2) an image configuration step of reconstructing an image from the two types of volume data obtained in the data acquisition step; (3) the image configuration step The obtained image is subjected to a coloring step of performing coloring processing such that the two types of qualitatively different images become different colors from each other, and (4) alignment processing of the two types of images obtained in the coloring step is performed. Synthetic step to overlap.
  • “qualityally different” means a state before and after administration of a specific condition (eg, Initial Spasm) or a drug occurs. This is because differences can be examined in detail by comparing qualitatively different states.
  • Volume data includes temporally and / or spatially different data.
  • images two-dimensional images or three-dimensional images can be employed. In the coloring step, different colors may be applied to each of the two types of images, or only one of them may be colored so that both may be displayed differently.
  • the open source software Image J National Institutes of Health (http://imagej.nih.gov/ij/)) can be used as software for alignment.
  • the method of screening a substance passing through the blood flow brain barrier in real time comprises: a non-human mammal, a fixing base for fixing the non-human mammal under anesthesia, and a surface of the non-human mammal's brain surface.
  • a screening apparatus comprising an imaging device capable of capturing a moving image with a two-photon laser microscope, characterized in that it is based on the moving image captured by the imaging device.
  • a cortical infarction model according to another invention is characterized by transient forebrain ischemia and unilateral carotid artery stenosis of a non-human mammal.
  • this cortical infarct model is used to treat the cortical brain infarct-formed brain so that it can be observed, and a two-photon laser microscope is used to acutely develop ischemia / abnormal contraction / reperfusion before cerebral infarction. It is possible to provide a method of observing non-human mammals with cerebral infarction which can observe temporally and spatially in real time temporally and spatially a subacute cerebral infarction pathological condition of cerebral infarction and cerebral infarction border area.
  • the method of administering the drug into the intracerebroventricularly is a method of administering the drug into the intracerebroventricular region using the non-human mammalian observation model of brain pathology in which the cranial craniotomy is provided from the outside of the cranium of the nonhuman mammal.
  • the tip of the cannula is located 0.5 mm posteriorly, 1.0 mm outward, 2.0 mm deep from the brain surface from the pregma Fixed at a predetermined position.
  • the drug When the drug is administered by dropping from the cranial craniotomy, there is a possibility that the drug may not reach the entire brain surface. Therefore, the drug can be distributed over the entire brain by detaining the insertion position of the cannula at a predetermined position using the method of the present invention. In addition, the above-mentioned predetermined position does not disturb the cannula even in observation using a microscope.
  • a light shielding device is used when observing an object using a microscope, and the inner peripheral diameter substantially equal to the outer peripheral diameter of the objective lens of the microscope, and the object It is characterized in that it comprises: a lower end edge contacting in a light blocking state; and when observing with the microscope, it is possible to block light from the side of the light blocking device.
  • the light shielding device of the present invention it is possible to substantially shield the observation site and the objective lens of the microscope, so that photographing can be performed even in dim light.
  • the light shielding device can also shield a slight amount of light, so that imaging with higher contrast can be performed. In addition, it has a preventive effect against accidents such as being turned on unexpectedly.
  • the present invention it is possible to provide a method for observing cerebral infarction diseased non-human mammals, a cerebral infarction diseased non-human mammal observation device, a data analysis method, a preventive / remedy drug for cerebral infarction and the like.
  • FIG. 1 It is the graph which expanded and displayed the blood flow change from immediately after two-sided common carotid artery occlusion to just before reopening in the part of the graph of FIG. Photograph obtained by two-dimensional laser blood flow measurement from the first stage blood flow reduction state after bilateral common carotid artery occlusion to the second stage blood flow reduction state (secondary CBF decline)
  • FIG. It is a graph which shows the relationship between the time from the bilateral common carotid artery occlusion to causing the blood flow reduction state at the second stage and the residual blood flow after the blood flow reduction at the first stage.
  • (D) is capillary from artery from volume data
  • Image figure which shows the result of having carried out 3D reconstruction so that it may be easy to understand vascular construction to a vein. It is a photograph figure which shows a result when a brain outer surface blood vessel is observed using the two-photon microscope of the bilateral carotid artery remote occlusion 20 minutes mouse model.
  • FIG. 7 is a photograph of Initial Spasms taken at high frame rates with a focus on the brain surface arteries and using a two-photon microscope to change magnification and scan sequence.
  • A shows the observation results of high magnification and high frame rate
  • B shows the observation results of low magnification and high frame rate
  • C shows the observation results of medium magnification and high frame rate.
  • Photograph (A) showing the change in blood vessel diameter over time from pre-infarction to reperfusion of each blood vessel in the pial artery (Pial Artery) on the surface of the brain and the penetrating artery (Penelating arteriole) in the deep brain
  • FIG. 16 is a photographic view showing the observation results of a deep cerebral capillary and a perforating artery using a two-photon microscope of a bilateral carotid artery remote occlusion 20-minute mouse model.
  • (A) is a photograph showing temporal changes with a depth of 200 ⁇ m to 300 ⁇ m
  • (B) is a magnified photograph of a capillary, respectively.
  • Two-photon photomicrograph (A), a magnified view (B) and a capillary showing an observation result obtained by enlarging the capillary and penetrating artery in the deep part of the brain in a bilateral remote carotid artery occlusion 20 min mouse model and photographing at a high frame rate
  • FIG. 6A is a graph (A) showing that the fluorescence intensity changes with time with bilateral carotid artery occlusion
  • FIG. 7B is a graph (B) showing that the fluctuation of the fluorescence intensity is different depending on the depth.
  • the graph (A) showing the relationship between the change in blood vessel diameter and the change in fluorescence intensity over time at a site at a depth of 200 ⁇ m to 300 ⁇ m in the bilateral carotid artery occlusion 20 min mouse model shows the change in penetrating artery and fluorescence signal intensity at each point It is a photograph figure (B).
  • (A) is a photograph showing extravasation before and after the first abnormal contraction of the pial surface artery of the brain
  • (B) is an enlarged photograph of (A)
  • (C) is the first abnormality around the capillary in the deep part of the brain
  • (D) is a graph showing the change in fluorescence intensity around blood vessels before and after the first abnormal contraction (left: around pial artery, right: around deep capillaries) .
  • wearing with a micro coil which were used for stricture lesion creation are each shown.
  • Magnetic resonance imaging (MRI) picture 24 hours after infarction in a cortical infarction model (A) and 2, 3,5-triphenyl tetrazolium chloride (TTC) stained picture (B) is shown.
  • (A) shows a full view of the craniotomy
  • FIG. 7 is a photographic view showing the structure and method of operation of the improved cannula. It is an image showing a reconstructed side image of volume data taken using a resonant scanner / GaAsP detector. (A) The result of imaging
  • A An image in which all arterial systems included in the VOI are emphasized and displayed
  • B an image in which all venous systems included in the VOI are emphasized and displayed. It is an image which shows the blood vessel construction connected to a vein from an artery through a capillary in VOI image
  • A is an image in which all of the artery A, the vein V, and the capillary C are highlighted
  • A shows a two-dimensional image before and after primary anomalous vasoconstriction in the deep VOI.
  • B shows the composite image which did not perform alignment processing, and the composite image which performed alignment processing.
  • C show abnormal vasoconstriction of arteries and veins captured from analysis of alignment composite images The three-dimensional processing method for the exhaustive analysis of the volume data acquired using resonant scanner * GaAsP PMT is shown.
  • A) shows a three-dimensional reconstructed image before and after the first abnormal blood vessel contraction in the deep part of VOI.
  • B shows the composite image which did not perform alignment processing, and the composite image which performed alignment processing.
  • (C) Deep brain image of composite image with and without alignment showing abnormal vasoconstriction of artery It is a two-photon photomicrograph (it acquired using resonant scanner * GaAsP PMT) which confirmed the plug formation (Plugging) of the blood cell by Initial Spasm.
  • the upper stage (A) shows the deep brain blood vessel, and the lower stage (B) shows an enlarged upper stage.
  • the states before Initial Spasm, during Initial Spasm, and after Initial Spasm are shown in order from the left in each stage.
  • Solid white arrows in the figure indicate penetrating arteries, and dotted white arrows indicate veins.
  • "*" Indicates a site where blood cells cause plugging. It is a photography figure which shows calcium imaging of cerebrovascular.
  • the upper part (A) shows the image of the brain surface
  • the lower part (B) shows the image of the deep brain. From the left side in each row, the states before Initial Spasm, the state where Initial Spasm has occurred, the state after Initial Spasm, and the state where Secondary Spasm have occurred are respectively shown.
  • the solid white arrows in the figure before Initial Spams indicate the cerebral surface arteries
  • the dotted white arrows indicate veins.
  • Thick arrows indicate increased fluorescence associated with arterial and venous spasms. The result of having conducted the test which confirms the spasm preventive effect of nicardipine is shown.
  • FIG. 5 is a photographic view of a three-dimensional reconstructed side view of the VOI.
  • the upper stage (A) shows an untreated case (No treatment), and the lower stage (B) shows an example of nicardipine administration.
  • the states of Initial Spasm, Initial Spasm start, and Initial Spasm propagation are shown in order from the left in each stage.
  • the upper side shows the brain surface
  • the lower side shows the deep brain.
  • the solid white arrows in the figure indicate the penetrating artery, and the dotted white arrows indicate the state of propagation of the Initial Spasm.
  • (A) The perspective photograph figure before mounting a light shielding device
  • (B) It is the perspective photograph figure after mounting a light shielding device.
  • the upper part (A) shows a photograph when the light shielding device is attached, the left side is a complete light shielding, the center is a general photographing environment, and the right side is a dim light environment.
  • the middle row (B) shows a photograph when the light shielding device is not attached, the left side is completely shielded, the center is a general photographing environment, and the right side is a dim light environment.
  • the lower part (C) shows an enlarged photograph, the left is an enlarged photograph of a general photographing environment without a light shielding device, and the right is an enlarged photograph of a dim light environment with a light shielding device attached.
  • FIG. 1 shows a carotid artery open / close device used to create a carotid artery remote occlusion model.
  • the device uses a commercially available cannula (20-G; inner diameter, 0.95 mm; length, 4 cm; Terumo Co., Ltd.) and nylon thread (3-0 polypropylene suture [PROLIN], length 12 cm, ETHICON, USA) (FIG. 1 (A), (B)).
  • FIG. 2 shows how the carotid artery open / close device for remote occlusion shown in FIG. 1 is attached to the mouse carotid artery, and the common carotid artery is actually occluded and reopened.
  • a median incision was made in the carotid artery of the mouse, and under a stereomicroscope, both common carotid arteries were dissected from the surrounding connective tissue, and a nylon thread was passed under the carotid arteries.
  • the two stumps were inserted together into a cannula and advanced until the stump of nylon thread came out on the other side.
  • FIGS. 2 (C) and 2 (D) indicate the carotid artery pulled into the nylon thread and pulled in half in the cannula.
  • FIG. 3 shows the results of blood flow changes measured using a two-dimensional laser blood flow meter using a mouse carotid artery remote occlusion model.
  • a two-dimensional laser flowmeter (Omegazone, Laser Speckle Blood Flow imager, Omegawave)
  • occlusion and recanalization of the common carotid artery are actually performed by the carotid artery open / close device.
  • Blood flow changes were measured. After anesthesia, the mouse head is fixed using a custom-made stereotactic device, and the skull is exposed by making a midline incision in the head, and a two-dimensional laser blood flow meter is observed from above the skull. Blood flow was measured at one shot per second.
  • FIG. 3 (A) the left common carotid artery is occluded (Left occlusion) after taking 5 minutes for a normal state (Pre) before occlusion, and 5 minutes later, the right common carotid artery Occluded (Bilateral occlusion: BCARO), and maintained the state of bilateral occlusion for 20 minutes, then recanalize bilaterally (Recanalization), and continuously performed up to 1 hour after reperfusion without moving the head observation area at all .
  • the first point is a point where blood flow decreases in two steps with bilateral occlusion (two step decreasing during BCARO).
  • FIG. 4 is an enlarged view of the blood flow change in the region of interest between immediately after bilateral common carotid artery occlusion and just before reopening. Of the 18 cerebral hemispheres, secondary cerebral blood flow reduction occurred within 6 minutes (dotted line and dotted arrow in FIG. 4) in 15 cerebral hemispheres. Arrows indicate 3 mice with second stage blood flow reduction that occurred more than 6 minutes after occlusion.
  • GFP green fluorescent protein
  • Intraperitoneal injection (8 ⁇ L / kg body weight) performed intravascular plasma (Plasma) staining, which resulted in GFP-positive astrocytes and pericytes in the brain parenchyma.
  • a cell neuropil can be confirmed as GFP weak positive, and a nerve cell can be confirmed as a shadow deficient cell in the neural net, and the blood vessel diameter can be confirmed by plasma staining with SR101.
  • platelets platelet
  • white blood cells leukocyte
  • red blood cells can be confirmed as a fluorescent shade in the SR101 Greekm staining.
  • FIG. 9 (A) shows a photograph of the unfolded head taken at a low magnification under a stereomicroscope, and the center shows a photograph of the entire unfolded head taken at a low magnification using a two-photon microscope.
  • SR101 fluorescence in red and GFP fluorescence in green it is possible to distinguish and confirm that the inside of the blood vessel is red and the brain parenchyma is green.
  • the upper dotted arrow in the center of FIG. 9A indicates the anterior cerebral artery
  • the lower solid arrow indicates the middle cerebral artery
  • the solid square indicates the boundary between the two arterial inner regions.
  • FIG. 9 (A) On the right of FIG. 9 (A), a magnified view of a photograph of the boundary area indicated by the solid line in the center of FIG. 9 (A) is shown. Arrows in the figure indicate the most peripheral branch (terminal branch) of the middle cerebral artery. The border region of the two blood vessels is the most peripheral region of the circulation, and in carotid artery occlusion, it is the most hemodynamically the site that is most susceptible to cortical infarction. Therefore, the volume of interest (VOI) is set in the boundary area so as to include the terminal branch of the middle cerebral artery (Fig. 9 (A), the dotted square on the right shows the brain surface of the VOI.
  • VOI volume of interest
  • FIG. 9 (B) shows the result of 3D reconstruction (Three-dimensional reconstructions) of volume data of each fluorescence. By performing 3D reconstruction, it is possible to observe any part of the VOI from any direction.
  • FIG. 9C shows the result of maximum intensity projection (MIP) processing of volume data. The left side shows the results of maximum intensity projection processing from the brain surface to a depth of 100 ⁇ m, the center from a depth of 100 ⁇ m to 200 ⁇ m, and the right side from a depth of 200 ⁇ m to 300 ⁇ m. Arrows indicate penetrating arteries running from the brain surface to the deep, and arrowheads indicate veins from the deep to the brain surface. In addition, by performing 3D reconstruction of volume data, observation at any cross section can be performed, and FIG.
  • FIG. 9D shows a state in which the entire traveling of the blood vessel traveling to the penetrating artery-capillary-vein is captured showed that.
  • the left side of FIG. 9D shows a 3D reconstruction weakly magnified image of volume data, the arrow shows an artery, and the arrowhead shows a vein.
  • the center shows a high magnification image. Arrows indicate arteries and arrowheads indicate veins.
  • a photograph is shown in which only the vasculatures selected from the center figure are extracted.
  • a blood vessel running from an artery to a vein through a capillary is shown by a dotted arrow.
  • the observation was performed in the same time course as the blood flow observation using a two-dimensional laser blood flow meter.
  • FIG. 3 (A) the imaging time for volume set per volume using a two-photon microscope requires 78.2 seconds, and the closure / resumption is synchronized with the timing of volume set imaging. went. From the time before the cerebral infarction to the relapse, 70 volumes of imaging were repeated continuously in the course of 91.3 minutes. Abnormal contraction was observed in the cerebrovascular in response to changes in blood flow using a laser Doppler flowmeter.
  • FIGS. 10 (A) and (C) show the change of the two-dimensional laser blood flow meter
  • FIGS. 10 (B) and (D) show the change of the blood vessel photographed by the corresponding two-photon microscope.
  • the solid arrows on the left side of FIG. 10B indicate arteries, and the dotted arrows indicate veins.
  • FIG. 10 shows a secondary abnormal contraction (Secondary Spasm).
  • the target was focused on the surface cerebral artery, the magnification was changed, and the scan sequence (Scan sequence) was changed to perform imaging at a high frame rate (interval) depth, 5 ⁇ m; thickness, 20 ⁇ m; slice number, 5; individual volume set scanning duration, 6.6 s).
  • FIG. 11A shows the observation results of high magnification and high frame rate.
  • each second indicates the number of seconds after the start of the first abnormal contraction (initial spasm onset)
  • the first abnormal contraction occurs at 4.6 ⁇ 3.7 min after bilateral common carotid artery occlusion (after BCARO onset)
  • the constriction resulted in 72% ⁇ 2% constriction as compared to that before abnormal contraction, and the duration of the constriction was 1.2 ⁇ 0.3 min.
  • FIG. 11 (B) shows the observation results of low magnification and high frame rate of the entire craniotomy.
  • FIG. 11B shows the dotted arrow indicates the anterior cerebral artery
  • the solid arrow indicates the middle cerebral artery
  • the dotted square indicates the border area between the two arteries.
  • the white arrow in the middle figure shows the outermost branch of the middle cerebral artery, which is the origin of abnormal contraction in the border area, and the arrowhead on the right, shows the abnormal contraction transmitted to the anterior cerebral artery and the middle cerebral artery central side .
  • FIG. 11C shows the observation results at a medium magnification and high frame rate.
  • the VOI includes the pial artery on the surface of the brain and the penetrating artery that vertically penetrates the brain parenchyma. It remains unclear whether there is a difference in vascular responsiveness (vessel diameter) at each site to ischemia. Therefore, the time-dependent changes from before the infarct to after reperfusion of each blood vessel diameter of the pial artery on the surface of the brain and the penetrating artery in the deep part of the brain (depth 150 ⁇ m and 300 ⁇ m) were measured.
  • FIG. 12A shows a 3D reconstruction image of the entire image from the pial artery to the penetrating artery included in the VOI.
  • the second row in FIG. 12 (A) shows the temporal changes of the pial artery, the third row the penetrating arteriole of 150 ⁇ m in depth, and the fourth row the penetrating arteriole of 300 ⁇ m in depth.
  • the FIG. 12 (B) graphically shows the change in blood vessel diameter at each blood vessel site.
  • each blood vessel is incomplete after the first abnormal contraction (initial spams), it causes re-expansion, but the degree is different, and re-expansion is better as in the pericardial surface of the brain, and as the penetrating artery in the deep brain, It was confirmed that the expansion would be bad. On the contrary, as for the change of the blood vessel diameter associated with the second abnormal contraction (secondary Spasm), the contraction tended to be stronger in the pial artery.
  • FIG. 13 (A) shows temporal change of an image in which the maximum intensity is projected to a depth of 200 to 300 ⁇ m in the VOI. Arrows in the figure indicate penetrating arterioles.
  • FIG. 13 (B) shows a magnified image of capillaries in the VOI.
  • FIG. 13 (B) shows a composite image (Merge) of SR101 and GFP, and the upper arrow indicates pericytes (pericite: GFP fluorescence positive) present in the capillary wall.
  • FIG. 14A shows a temporal change after the maximum intensity projection processing. Arrows in the figure indicate penetrating arterioles in which the first abnormal contraction occurred, and arrowheads indicate penetrating arteries in which partial re-dilation has occurred.
  • FIG. 14 (B) shows an enlarged image of a dotted square part in the upper left side of FIG. 14 (A).
  • the left side of FIG. 14B shows a composite image (Merge) of SR101 and GFP, and the arrow shows pericytes (GFP positive) present in the capillary wall. It was observed that the capillary lumen was completely occluded in the vicinity of the pericyte-present area along with the abnormal contraction (FIG. 14 (B) right view).
  • FIG. 14 (B) shows an enlarged image of a dotted square part in the upper left side of FIG. 14 (A).
  • the left side of FIG. 14B shows a composite image (Merge) of SR101 and GFP, and the arrow shows pericytes (GFP positive) present in the capillary wall. It was observed that the capillary lumen was completely occluded in the vicinity of the pericyte-present area along with the abnormal contraction (FIG. 14 (B) right view).
  • FIG. 14 (C) shows a time-dependent change in blood vessel diameter in capillaries at a site where there is no total occlusion (pericyte is not present). At the site where pericytes were not present, the capillaries did not have a total occlusion, and this condition was observed to be continued from the propagation of the first abnormal contraction of the artery until reperfusion. .
  • FIG. 15 (A) Before the occlusion in FIG. 15 (A), there is almost no space between cells such as endothelial cells around the capillaries, the basement membrane (blood-brain barrier), pericytes, and glial cells.
  • FIG. 15 (B) after 20 minutes of bilateral remote carotid artery occlusion, the pericytes are in a state of tightening the capillaries, and as a result, the lumen region of the capillaries is retracted and rupture of the glial cell process is observed.
  • FIG. 16 shows the temporal change of the maximum intensity projection processed slice having a depth of 200 to 300 ⁇ m (arrows indicate penetrating arteries).
  • VOI was divided into depths of 0 to 100 ⁇ m, 100 to 200 ⁇ m, and 200 to 300 ⁇ m, and changes in GFP fluorescence signal intensity over the entire screen at each depth were shown.
  • changes in signal intensity and changes in blood vessel diameter of penetrating arteries were examined in detail at a site at a depth of 200 to 300 ⁇ m.
  • the fluorescence signal increased sharply and decreased sharply in synchronization with the first abnormal contraction (first point).
  • FIG. 17A shows the relationship between the change in blood vessel diameter and the change in signal intensity over time at a site with a depth of 200 to 300 ⁇ m. A dramatic change in fluorescence signal intensity was observed at 3 points in synchronization with the change in blood vessel diameter.
  • FIG. 17B exemplifies an image showing a change in blood vessel diameter of a penetrating arteriole at each point and a change in fluorescence signal intensity (an arrow indicates a penetrating artery).
  • FIG. 18A shows a time course of carotid artery occlusion / reopening and imaging.
  • FIG. 18 (B) arrow Unlike the second abnormal contraction associated with the 20-minute occlusion, a high degree of vasoconstriction was observed from the peripheral side to the central side of the artery (FIG. 18 (B) arrow). Of the 4 mice with complete arrest of blood flow and abnormal contraction, 2 died before reperfusion and 2 died early in reperfusion. In one patient who survived to reperfusion, partial fusiform vasodilation (Fig. 18 (B) arrowhead) was observed in the blood vessels along with reperfusion, but complete blood flow resumption was not observed (Fig. 18 (B)). The remaining case showed vasodilation immediately after, but the blood flow decreased again within 15 minutes and then died (FIG. 18 (C)).
  • the first pathway is a pathway in which Ca 2+, which flows in from outside the cell through a voltage-gated Ca 2+ channel, binds to calmodulin and activates myosin light chain kinase.
  • the second pathway is intracellular binding by binding adrenergic noradrenaline released from the nerve terminal with activation of the perivascular sympathetic nerve to the ⁇ -adrenergic receptor.
  • Ca 2+ is released from sarcoplasmic reticulum, and it binds to calmodulin and activates myosin light chain kinase.
  • Rho-associated coiled forming kinase suppresses the activity of myosin light chain phosphatase and suppresses dephosphorylation of myosin light chain
  • ⁇ -adrenergic receptor antagonist ⁇ -adrenergic receptor antagonist
  • phentolamine mesylate a ROCK inhibitor
  • ROCK inhibitor a ROCK inhibitor
  • the second step was direct administration of the drug from the brain surface and direct observation using a two-photon microscope.
  • the condition of the blood vessel from before the blood vessel occlusion to 20 minutes after the bilateral carotid artery remote occlusion was continuously observed to confirm the preventive effect of each drug on the primary abnormal contraction.
  • the fasudil hydrochloride-administered group the same primary abnormal contraction as in the control group (untreated; upper in FIG. 21) was observed (FIG. 21 middle).
  • the phentolamine mesylate administration group it was confirmed that the primary abnormal contraction can be
  • Fasudil hydrochloride did not suppress abnormal contraction and died in both cases.
  • suppression of the first abnormal contraction and the second abnormal contraction was recognized in all cases, and awakening from anesthesia was recognized.
  • FIG. 22 in the phentolamine mesylate administration group, both the first abnormal contraction and the second abnormal contraction were suppressed, and the blood flow was maintained until just before reopening. In addition, along with recanalization, good vasodilation and blood flow recovery were observed.
  • prevention of abnormal contraction can be a new treatment method for cerebral infarction, that abnormal contraction can be suppressed by blocking ⁇ -adrenoceptor, Phentramine mesylate is abnormal contraction as one of the drugs. It has been confirmed that the observation method of the present embodiment is effective as a method for determining the efficacy of a drug for preventing / treating abnormal contraction, which can be prevented.
  • Stenotic lesions may be completely occluded by silk thread ligation, or microcoil (micro coil: using 0.22 mm, 0.20 mm, 0.18 mm, 0.16 mm coil, the degree of stenosis can be changed by changing the coil inner diameter)
  • a stenotic lesion was created by mounting the S from the outside.
  • FIG. 23A shows the form of the micro coil actually used.
  • FIG. 23 (B) shows a schema (schematic) in which a micro coil is attached to a carotid artery
  • FIG. 23 (C) shows a photograph of the micro coil attached to a mouse carotid artery.
  • the size of the infarct could be controlled based on the degree of stenosis (diameter of the coil used).
  • cortical infarction can be evaluated by MRI, TTC staining or the like which is a general cerebral infarction evaluation method.
  • FIG. 24 (A) shows the evaluation results of infarcts after 24 hours using MRI (DWI), and FIG. 24 (B) shows the evaluation results of infarcts after 24 hours by TTC staining.
  • MRI high signal
  • TTC the white unstained part indicates an infarct.
  • the first feature of the model of the present embodiment is that the ischemic boundary area can be directly observed using a two-photon microscope. In the subacute phase of cerebral infarction, it is divided pathophysiologically into the ischemic central area and its surrounding area. The central portion of the ischemia is a site which has already fallen into necrosis and is a site which can not be rescued even by drug administration.
  • the area surrounding the ischemia is a site where cell death has not yet occurred, but necrosis may occur as time passes.
  • cerebral infarction takes over about 3 days. Therefore, for the treatment of the subacute stage, many drugs are focused on how to help this area around the ischemia.
  • the model developed by the present inventor which combines transient forebrain ischemia and a one-sided carotid artery stenosis, and the observation method using a two-photon microscope, directly observes the central area of ischemia and the peripheral area of ischemia.
  • the main seat of the ischemic central area is mainly formed in the border area of the anterior cerebral artery and the middle cerebral artery, and the peripheral area of the ischemia is surrounded. Therefore, by creating a cranial craniotomy in the center of the parietal lobe area, the ischemic peripheral area formed at the border between the anterior cerebral artery and the middle cerebral artery area can be directly observed using a two-photon microscope.
  • FIG. 25 shows the observation results of the boundary region with a two-photon microscope.
  • FIG. 25 (A) shows a complete image of the cranial craniotomy.
  • the left side of the dotted line shows the survival region where the fluorescence is retained, and the right side of the dotted line shows the region where the fluorescence is reduced and the necrosis is caused.
  • FIG. 25 (B) shows an enlarged view of the boundary area.
  • the left figure is a picture showing blood vessels (SR101), the right figure is a picture showing brain parenchyma (GFP).
  • the left side of the dotted line indicates the non-infarcted ischemic area, and the right side of the dotted line indicates the infarct.
  • the phenomenon in which the leukocytes that have extravasated leaked actively act in the border area (the upper part of the dotted line shown in FIG. 26A shows the ischemic peripheral area and the lower part shows the infarct. Arrows in the figure indicate leukocytes leaked out of the blood vessel, and it was observed that the leukocytes were actively moving by reproducing the moving image).
  • the brain edema in the central portion of the ischemia a phenomenon in which the peripheral area of the ischemia was pressed and compressed (arrows in FIG.
  • the inflammatory reaction caused by edema and white blood cell activity is recognized as a mechanism of exacerbation of cerebral infarction, and the method of the present embodiment for directly evaluating the actual state thereof is a drug for the purpose of salvaging the peripheral area of ischemia. It is optimal as a method of drug efficacy evaluation.
  • a through hole 101 penetrating the wall surface of the cannula 100 is formed at an arbitrary position (3 mm in this embodiment) from the tip of the cannula 100 (FIG. 27A).
  • the through holes 101 are provided in the vertical direction of the cannula 100.
  • the silk thread 102 is inserted through the two through holes 101 (FIG. 27B).
  • the silk thread 102 is tied and the excess portion is cut away to form the stopper 103 (FIG. 27 (C)).
  • a blocker (a nylon thread 104 in this embodiment) for blocking the carotid artery is inserted into the inside of the cannula 100.
  • the nylon thread 104 is inserted at its both ends (not shown) from the side of the opening 100A at which the stopper 103 is provided in the cannula 100 so that it can be manipulated from other openings (not shown) (FIG. D)).
  • the carotid artery can be inserted through the locking ring 105 formed at the center of the nylon thread 104.
  • the nylon thread 104 is inserted into the inside of the cannula 100 so as to position the carotid artery inside the locking ring 105.
  • the projecting position of the locking ring 105 is pulled toward the cannula 100 (FIG. 27 (E)). Since the projection position of the locking ring 105 is regulated by the position of the stopper 103, the blocking state of the blood flow of the blood vessel is prescribed at a predetermined position (FIG. 27 (F)).
  • the blood flow blocking state of the carotid artery can be defined in advance, and this state can be implemented with good reproducibility, so that it is possible to provide a cannula that can perform experiments under constant conditions.
  • ⁇ New shooting method> There is a shooting speed as a problem when acquiring the VOI. A longer imaging time is required to obtain higher resolution images.
  • a galvano scanner operated by a galvano motor as the scanner and an alkali detector detector using an alkali metal photomultiplier tube (photomultiplier (PMT)) as the detector
  • PMT photomultiplier
  • a brain of 300 ⁇ m from the surface The imaging time of 82 seconds was required to slice at 5 ⁇ m intervals to the deep part and perform 61 imaging.
  • 3D re-synthesis was performed based on the acquired image, since the imaging slice interval was 5 ⁇ m, a part of the capillary blood vessel between slices became in a state of missing (FIG. 28 (A)).
  • GaAsP gallium arsenide phosphide
  • Resonant scanners scan mirrors with resonant motion, so they can scan faster than galvano scanners.
  • the GaAsP external detector can detect finer fluorescence at a high S / N ratio as compared to the alkali detector, so that a clearer image can be obtained even when the output of the excitation laser is reduced.
  • FIG. 28 (B) When 3D recomposition was performed based on the acquired image, a clearer image could be acquired without missing images (FIG. 28 (B)).
  • This method it was possible to obtain volume data at high resolution and at high speed to the deep brain. It was also possible to analyze the travel of all blood vessels included in the VOI, which was difficult with the conventional method. All arterial and venous systems included in the VOI were identified (Fig. 29 (A), (B)). In addition, the entire volume of the blood vessel running from the artery (A) to the vein (V) via the capillary (C) could be easily analyzed (FIGS. 30 (A), (B), (C)).
  • the transition site of arteries and capillaries the transition site from capillaries to veins, etc.
  • the ambiguity is large including the definition, and in single slice analysis, the arterioles, capillaries, and venules are separated. Was difficult. These can be easily determined by using the method of the present embodiment.
  • observation of a pathological condition of each blood vessel site, evaluation of efficacy of each blood vessel site, and the like can be performed.
  • the acquired volume data includes a large amount of images.
  • the imaging method using a resonant scanner and a cooled high sensitivity gallium arsenide phosphide (GaAsP) external detector in 75 minutes of continuous imaging, the image will be close to 20,000 images, and all changes can be identified simply by visual inspection. It is impossible to do. Therefore, we have established an analysis method to capture minor morphological changes efficiently.
  • FIG. 31 (A) shows a two-dimensional image of the deep brain region before and after Spasm (solid arrows indicate arteries, dotted arrows indicate veins).
  • FIG. 31 (B) shows a superimposed image with and without rigid body registration.
  • the result is a composite image in which a shift occurs, and evaluation is difficult, but by performing the alignment (right side), the shift can be almost corrected.
  • Analysis results using this method show that after Initial Spasm, contraction continues in capillaries before leading to arteries, veins and veins.
  • FIG. 31 (C) shows a magnified image of a superimposed image of artery and vein.
  • the overlapping part of the two images (blood vessels after Spasm), and the outside (arrows) indicate the morphology of blood vessels before Spasm. It indicates that vasoconstriction is sustained even in the deep brain region of arteries and veins and after Initial Spasm.
  • FIG. 32A shows three-dimensional reconstruction images of volume data before and after Spasm. If movement of the brain causes a gap between volume data, use the open source software Image J (National Institutes of Health (http://imagej.nih.gov/ij/)) to Alignment was done. By creating composite data of two volume data in the same manner as a two-dimensional image, it was possible to observe the inside of the volume three-dimensionally from any direction.
  • Image J National Institutes of Health
  • FIG. 32 (B) shows a three-dimensional reconstruction image of superimposed volume data according to the presence / absence of alignment (the left side indicates “no alignment” and the right side indicates “alignment” (also in FIG. 32C). the same).).
  • FIG. 32 (C) shows a magnified image of the deep brain with or without alignment (arrows indicate arteries). The alignment corrected the deviation, and it was found that the stenosis in the deep brain artery continued even after Initial Spasm.
  • the two-dimensional analysis method is characterized in that it can be easily performed in a short time.
  • the three-dimensional analysis method requires a large amount of volume data processing, so processing takes time, but all changes within the VOI Is characterized in that it can be observed comprehensively.
  • a calcium imaging method for vascular smooth muscle has not been established.
  • As the mechanism of vasoconstriction the existence of various mechanisms such as the influx of Ca from extracellular, the intracellular efflux of stored Ca from intracellular endoplasmic reticulum, the increase of Ca sensitivity, etc. have been reported.
  • Fluo 4-AM green fluorescence
  • Pluronic F-127 20% solution in DMSO a marked increase in green fluorescence intensity was observed in smooth muscle cells in the surface blood vessels of the surface arterioles and veins with Initial Spasm (FIG. 34) (A) thick arrows). This fluorescence increase persisted after re-expansion, and a further increase in fluorescence intensity was observed as Secondary Spasm started.
  • nicardipine a voltage-dependent calcium channel blocker inhibitor
  • the light shielding device 201 will be described with reference to FIG.
  • the light shielding device 201 is formed of, for example, a black plastic in a cylindrical shape communicating in the vertical direction.
  • the light blocking device 201 blocks external light from the side so as not to leak into the cylinder.
  • the inner circumferential diameter of the opening 202 of the light shielding device 201 is set substantially equal to the outer circumferential diameter of the objective lens 204 of the microscope.
  • the lower edge 203 of the light shielding device 201 can perform observation under a microscope in a state of being in contact with the object in a light shielding state. As shown in FIG. 37, the light shielding device 201 is attached to the lower end side of the objective lens 204 of the microscope, and observation with a microscope is performed in a state where the lower end edge 203 is in close contact with the object.
  • FIG. 38 shows a comparison based on the presence or absence of the light shielding device 201.
  • "Completely blocked” covers the microscope with a shaded curtain and shows the condition in a completely dark place (the left side of FIGS. 38A and 38B).
  • “General shooting environment” indicates a state in which light from a monitor screen of a microscope or a start lamp of various devices is leaking although it is in a dark place (generally, a condition under which shooting is performed.
  • FIG. Center of B) and left of (C) “Lighting environment” indicates that the A4 document can read 12-point characters and the remote control in the observation target is bright enough (see FIG. 38 (A), (B) on the right side and Right of C)).
  • FIG. 38 (A), (B) on the right side and Right of C) In the mounted state of the device (right side in FIGS.
  • the observation site and the objective lens 204 of the microscope can be substantially shielded, so that photographing can be performed even in dim light. Since the light shielding device 201 can also shield a slight amount of light, it is possible to perform imaging with better contrast. In addition, it has a preventive effect against accidents such as being turned on unexpectedly. Moreover, this light shielding device 201 can be applied not only to a two-photon microscope but also to observation using a general confocal microscope or a fluorescence microscope.
  • the present embodiment it is possible to provide a method for observing cerebral infarction diseased non-human mammals, a cerebral infarction diseased non-human mammal observation device, a preventive and / or a remedy for cerebral infarction, and the like.

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Abstract

Le problème décrit par la présente invention est de proposer un procédé d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain, et un dispositif d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain. La solution du problème selon la présente invention consiste en un procédé d'observation d'un mammifère non humain en état d'infarctus cérébral, le procédé comprenant la mise en œuvre d'une intervention pour rendre le cerveau d'un mammifère non humain anesthésié observable en utilisant un modèle d'occlusion bilatéral distant de l'artère carotide commune pour le mammifère non humain, et l'observation, dans le temps/dans l'espace et en temps réel, de la pathologie de l'infarctus cérébral depuis avant l'apparition d'un infarctus dans un vaisseau sanguin cérébral jusqu'à l'ischémie/une contraction anormale/la reperfusion en utilisant un microscope à balayage laser à deux photons.
PCT/JP2018/030683 2017-08-21 2018-08-20 Procédé d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain, et dispositif d'observation utilisant un modèle d'infarctus cérébral de mammifère non humain Ceased WO2019039438A1 (fr)

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