CN113383750A - 一种进展性缺血性脑卒中模型的构建方法 - Google Patents
一种进展性缺血性脑卒中模型的构建方法 Download PDFInfo
- Publication number
- CN113383750A CN113383750A CN202110769449.4A CN202110769449A CN113383750A CN 113383750 A CN113383750 A CN 113383750A CN 202110769449 A CN202110769449 A CN 202110769449A CN 113383750 A CN113383750 A CN 113383750A
- Authority
- CN
- China
- Prior art keywords
- light source
- progressive
- ischemic stroke
- mouse
- stroke
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000000750 progressive effect Effects 0.000 title claims abstract description 42
- 208000032382 Ischaemic stroke Diseases 0.000 title claims abstract description 30
- 238000010276 construction Methods 0.000 title claims abstract description 9
- 241001465754 Metazoa Species 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 19
- 208000005189 Embolism Diseases 0.000 claims abstract description 9
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 33
- 210000004556 brain Anatomy 0.000 claims description 13
- 241000699670 Mus sp. Species 0.000 claims description 12
- 210000000683 abdominal cavity Anatomy 0.000 claims description 10
- IICCLYANAQEHCI-UHFFFAOYSA-N 4,5,6,7-tetrachloro-3',6'-dihydroxy-2',4',5',7'-tetraiodospiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 IICCLYANAQEHCI-UHFFFAOYSA-N 0.000 claims description 7
- 230000001678 irradiating effect Effects 0.000 claims description 7
- 229930187593 rose bengal Natural products 0.000 claims description 7
- 229940081623 rose bengal Drugs 0.000 claims description 7
- STRXNPAVPKGJQR-UHFFFAOYSA-N rose bengal A Natural products O1C(=O)C(C(=CC=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 STRXNPAVPKGJQR-UHFFFAOYSA-N 0.000 claims description 7
- 210000003625 skull Anatomy 0.000 claims description 6
- 229930182555 Penicillin Natural products 0.000 claims description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 5
- 229910052736 halogen Inorganic materials 0.000 claims description 5
- 150000002367 halogens Chemical class 0.000 claims description 5
- 229940049954 penicillin Drugs 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 206010002091 Anaesthesia Diseases 0.000 claims description 4
- 230000037005 anaesthesia Effects 0.000 claims description 4
- 230000036760 body temperature Effects 0.000 claims description 4
- 210000000337 motor cortex Anatomy 0.000 claims description 3
- 229960002078 sevoflurane Drugs 0.000 claims description 3
- DFEYYRMXOJXZRJ-UHFFFAOYSA-N sevoflurane Chemical compound FCOC(C(F)(F)F)C(F)(F)F DFEYYRMXOJXZRJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000009395 breeding Methods 0.000 claims 1
- 230000001488 breeding effect Effects 0.000 claims 1
- 208000006011 Stroke Diseases 0.000 abstract description 23
- 230000001575 pathological effect Effects 0.000 abstract description 9
- 230000002490 cerebral effect Effects 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 7
- 206010008190 Cerebrovascular accident Diseases 0.000 abstract description 6
- 208000027418 Wounds and injury Diseases 0.000 abstract description 5
- 230000007971 neurological deficit Effects 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 4
- 208000037906 ischaemic injury Diseases 0.000 abstract description 3
- 208000037273 Pathologic Processes Diseases 0.000 abstract description 2
- 206010043647 Thrombotic Stroke Diseases 0.000 abstract description 2
- 238000010171 animal model Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 239000002547 new drug Substances 0.000 abstract description 2
- 230000009054 pathological process Effects 0.000 abstract description 2
- 230000002085 persistent effect Effects 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 238000011631 stroke animal model Methods 0.000 abstract description 2
- 229940126585 therapeutic drug Drugs 0.000 abstract description 2
- 238000010837 poor prognosis Methods 0.000 abstract 1
- 230000006870 function Effects 0.000 description 14
- 206010008118 cerebral infarction Diseases 0.000 description 12
- 210000005036 nerve Anatomy 0.000 description 12
- 238000000465 moulding Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000001154 acute effect Effects 0.000 description 7
- 208000026106 cerebrovascular disease Diseases 0.000 description 7
- 230000000302 ischemic effect Effects 0.000 description 7
- 230000002025 microglial effect Effects 0.000 description 7
- 210000005013 brain tissue Anatomy 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 208000028867 ischemia Diseases 0.000 description 5
- 210000000274 microglia Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 201000006474 Brain Ischemia Diseases 0.000 description 4
- 206010008120 Cerebral ischaemia Diseases 0.000 description 4
- 230000003111 delayed effect Effects 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 229950003937 tolonium Drugs 0.000 description 4
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000005452 bending Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 208000029028 brain injury Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000009193 crawling Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000003194 forelimb Anatomy 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 230000004770 neurodegeneration Effects 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 101001092197 Homo sapiens RNA binding protein fox-1 homolog 3 Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 102100035530 RNA binding protein fox-1 homolog 3 Human genes 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 108010084652 homeobox protein PITX1 Proteins 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 210000003657 middle cerebral artery Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 230000016273 neuron death Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 206010070670 Limb asymmetry Diseases 0.000 description 1
- 101100246038 Mus musculus Ptpn5 gene Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 210000001642 activated microglia Anatomy 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000010102 embolization Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000036732 histological change Effects 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000006724 microglial activation Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007659 motor function Effects 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- 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
- 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
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- 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
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
-
- 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
- A01K2207/00—Modified animals
- A01K2207/35—Animals modified by environmental factors, e.g. temperature, O2
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0375—Animal model for cardiovascular diseases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了进展性缺血性脑卒中模型的构建方法。进展性缺血性脑卒中是一种特殊类型的卒中,表现为神经功能缺损持续加重,预后差,缺乏有效药物,临床治疗非常棘手。由于缺少能够准确模拟进展性卒中病理过程的动物模型,导致该病的病理机制研究和治疗药物研发均受到较大局限。本发明通过改进光化学栓塞法的相关实验参数,使动物的脑缺血损伤程度符合进展性卒中的病理特点,成功构建了进展性缺血性脑卒中模型。本发明的有益效果主要体现在:提供了一种小鼠进展性缺血性脑卒中模型的新造模方法,填补了学术界进展性缺血性脑卒中动物模型的空白。同时,手术简便易行,动物创伤小,重复性好,且接近人类血栓性脑卒中的发病过程,对该病的病理机制研究和新药新靶点研发具有重要的应用价值。
Description
(一)技术领域
本发明涉及一种进展性缺血性脑卒中模型的构建方法。
(二)背景技术
缺血性脑卒中是一种严重的神经系统疾病,已经成为我国主要的致死和致残病因。进展性脑卒中(progressive ischemic stroke,PIS)是缺血性脑卒中的一个特殊类型,指脑卒中虽经临床积极干预,但导致脑缺血的原发病理过程仍继续进展,发病数天至一周内,出现神经功能缺损逐渐进展或阶梯式加重的一类脑梗死,约占缺血性脑卒中的30%。相比其它脑卒中类型,进展性脑卒中患者致残致死率更高,预后更差,神经功能缺损更严重,一旦发生,仅以对症治疗为主,是临床较难控制的卒中类型。由于病人入院后症状加重,常引起医疗纠纷。因此,亟需探明进展性脑卒中的关键发病机制,并寻找有效的药物干预方法。
相比于其他类型的脑卒中,进展性脑卒中的临床与基础研究较少且滞后,其主要原因与缺乏精准有效的动物研究模型有关。经典的线栓模型、光化学栓塞模型和大鼠脑血栓栓塞模型主要模拟的均是急性缺血性脑损伤的常规发病过程,并不能准确反映出进展性卒中的病理变化特征——脑梗死后一周左右患者神经功能会出现标志性的二次恶化现象,故不能直接应用于进展性卒中的研究,亟需构建新型针对进展性卒中的动物模型,以推动该类疾病的病理机制研究及相关治疗药物的研发进程。
(三)发明内容
本发明目的是一种进展性缺血性脑卒中模型的构建方法。
本发明采用的技术方案是:
一种进展性缺血性脑卒中模型的构建方法,其特征在于所述方法是采用光化学栓塞法,使用的冷光源可见光发光直径为2.0~2.5mm,光源垂直贴于颅骨上方,并使光源圆心较前卤向右偏移1.0~1.5mm;先腹腔注射玫瑰红溶液,剂量为80~100mg/kg,3~5min后进行冷光源照射10~15min,冷光源强度为150000~16000lux,照射结束后后移除冷光源,缝合切口并消毒,腹腔注射青霉素,剂量为2~5万U/kg,将动物置于恒温平板上至其苏醒体温恢复正常,并放回笼内继续饲养14天以上,获得进展性缺血性脑卒中模型。
光化学栓塞法的常规做法:采用固定波长光源,通常为波长560nm的光束,光照直径5.0mm左右,照射大脑中动脉起始端,时长20min,主要模拟的是急性局灶性大脑中动脉栓塞(Middle cerebral artery occlusion,MCAO)。MCAO是最经典的急性脑缺血模型,缺血损伤较重,表现为动物脑梗死体积大,神经功能评分较低,但缺血后期并无损伤持续加重的现象,所以明显区别于进展性卒中的疾病特点。本发明对经典光化学栓塞法的实验参数进行了重点优化和改进:首先,照射脑区选择了运动皮层,相比于大脑中动脉起始端,照射该脑区会针对性损伤动物的运动功能,而对其他脑区和功能的影响较小,能更好地模拟进展性卒中的疾病特点;其次,通过反复摸索实验条件,最终将光照直径缩小至2.0mm,照射时间缩短至15min,可大大减轻动物的急性期损伤,表现为动物的脑梗死体积更小,死亡率更低,模型稳定性更好,更有利于对缺血后期神经功能和组织学改变的研究(进展性卒中的关键病理特征主要发生于缺血后期);再次,所用的冷光源优化为非固定波长可见光光源,并经反复试验,最终确定了最优化的光照强度,相比于固定波长光源,最大的优势是实验成本降低,设备可及性高。
具体的,所述方法包括如下顺序步骤:
(1)选取10~12周龄、体重23~25g的雄性小鼠,七氟烷气体维持麻醉;
(2)将小鼠头部固定于脑立体定位仪上,剔除头部毛发并表面消毒,通过正中切口剪开皮肤,暴露前囟;
(3)冷光源发光直径为2.0mm,光源垂直贴于颅骨上方,并使光源圆心较前卤向右偏移1.5mm;
(4)先腹腔注射玫瑰红,剂量为100mg/kg,5min后进行冷光源照射15
min,冷光源强度为16000lux;
(5)照射结束后后移除冷光源,缝合切口并消毒,腹腔注射青霉素,剂量为5万U/kg;
(6)将动物置于恒温平板上至其苏醒体温恢复正常,并放回笼内继续饲养14天,获得进展性缺血性脑卒中模型。
建模所用小鼠可以是各种种类小鼠,如各种品系的野生型和基因突变型小鼠等。优选的,所述小鼠为C57BL/6J野生型雄性小鼠。
具体的,步骤(4)中冷光源照射的脑区位于运动皮层。
本发明优化了光化学栓塞法的实验参数,包括动物的周龄及体重;光源直径和强度、照射脑区位置及照射时间等,通过腹腔注射玫瑰红溶液(100mg/kg)诱导血栓,引发小鼠大脑皮层局灶性缺血。造模后14天内每天通过检测小鼠的网格错步率和圆桶不对称趴壁率,评价小鼠的感觉运动神经功能,可观察到光化学栓塞后1天小鼠神经功能损伤,而从光化学栓塞后第4~7天,其神经功能呈进展性恶化,反映了进展性缺血性脑卒中的神经功能变化特点,也符合临床观察到的进展性卒中常见于卒中后一周发作的规律。此外,通过免疫组织病理切片染色发现,本模型小鼠的病理基础不同于普通急性脑缺血损伤模型,其神经功能缺损与小胶质细胞的延迟激活有关,而与神经元丢失无关,表明新型小鼠进展性脑卒中模型构建成功。
本发明的有益效果主要体现在:发明提供了一种小鼠进展性缺血性脑卒中模型的新造模方法,填补了学术界进展性缺血性脑卒中动物模型的空白。同时,手术简便易行,动物创伤小,重复性好,且接近人类血栓性脑卒中的发病过程,对该病的病理机制研究和新药新靶点研发具有重要的应用价值。
(四)附图说明
图1为小鼠进展性缺血性脑卒中模型的实验结果图(n=8),左图为小鼠手术前腹腔注射100mg/kg的玫瑰红溶液,PT后14天甲苯胺蓝染色的脑梗死体积照片,右图为脑梗死体积柱形图。
图2为进展性缺血性脑卒中小鼠造模后14天内神经功能缺损进展情况(n=8-10),每天测试其(A)网格爬行错步率;(B)圆筒实验趴壁不对称率,以反映其神经功能缺损情况。##P<0.01v.s造模后1天,###P<0.001v.s造模后1天,*P<0.05v.s造模后3天,***P<0.001v.s造模后3天。
图3小鼠造模后1天及7天检查梗死半暗带脑区存活神经元密度。(A-C)假手术、造模后1天、造模后7天神经元NeuN染色情况,组织免疫染色局部放大图分别显示在(D-F)。(G)对上述组别的存活神经元密度进行统计分析(n=6)。
图4小鼠造模后不同时间点检查梗死半暗带脑区小胶质细胞数量及形态。(A-E),分别为假手术组,造模后1天、4天、7天、14天的代表性图片。(F)围绕梗死区做半径200微米的环形套圈,观察不同距离内小胶质细胞数量,可见造模后4-7天梗死区周边小胶质细胞显著增加。(G-K)分别为上述组别免疫荧光染色局部放大的代表性图片,分别对激活小胶质细胞的形态(L)充实度和(M)坚固性进行半定量分析,可见梗死后4-7天小胶质细胞延迟激活。
(五)具体实施方式
通过下列实例对本发明的内容做进一步说明,但本发明的保护范围,不限于此。
实施例1:构建光化学栓塞致进展性缺血性脑卒中的小鼠模型
(1)选取10~12周龄、体重23~25g的C57BL/6J野生型雄性小鼠,七氟烷气体维持麻醉;
(2)将小鼠头部固定于脑立体定位仪上,剔除头部毛发并表面消毒,通过正中切口剪开皮肤,暴露前囟,将一直径2.0mm的光导纤维固定紧贴在颅骨上方;
(3)冷光源发光直径为2.0mm,光源垂直贴于颅骨上方,并使光源圆心较前卤向右偏移1.5mm;
(4)先腹腔注射玫瑰红,剂量为100mg/kg,5min后进行冷光源照射15min,冷光源强度为16000lux;
(5)照射结束后后移除冷光源,缝合切口并消毒,腹腔注射青霉素,剂量为5万U/kg。
(6)将动物置于恒温平板上至其苏醒体温恢复正常,并放回笼内继续饲养14天。
小鼠冰冻脑组织切片制备:
小鼠注射过量戊巴比妥钠麻醉安乐死,用4℃0.9%的生理盐水和4%的多聚甲醛进行心脏灌流,取脑后,将脑组织浸泡于4%的多聚甲醛中4℃过夜,之后用新鲜配制的30%的蔗糖溶液浸泡脱水48h,最后用冰冻切片机对小鼠脑组织进行25mm厚度的冠状面冰冻切片。对于甲苯胺蓝染色的组织切片,用1×PBS漂洗3次,以用1%甲苯胺蓝溶液浸染20分钟,清水漂洗3次。经75%、85%、95%乙醇以及无水乙醇梯度漂洗后晾干,二甲苯透明化5分钟,明场显微镜观察。
造模14天后,甲苯胺蓝染色检测脑梗死体积。结果发现,该模型小鼠的脑梗死体积为0.8±0.05mm3(图1)。
实施例2:进展性缺血性脑卒中小鼠造模后14天内神经功能缺损进展情况
小鼠错步试验:
将小鼠置于40cm边长、内部1cm见方的网格上,网格由直径1mm的铁丝制成,离地高度30cm。网格底部设置一摄像头,拍摄小鼠在网格上的自由活动,正常小鼠自由运动时前后足均踩踏于铁丝上,由于脑缺血影响,缺血脑对侧前后足踏空网格,每踏空一步记为错步一次。拍摄小鼠自由活动总步数100步,记录错误步数并计算错步率(图2A)。
圆筒试验:
将小鼠放在一个直径10cm,高度15cm的透明圆筒中,在圆筒的正上方放置一个摄像头,用来记录小鼠的自由活动。当小鼠站立触碰筒壁时,分别记录5分钟内小鼠左侧,右侧或者左右前肢同时触碰筒壁的时间(图2B)。
小鼠肢体不对称率=(未损伤前肢触碰时间-损伤前肢触碰时间)/总触碰时间×100。
按所述方法构建小鼠进展性卒中模型,可观察到小鼠在造模后第1天即出现神经功能损伤,表现为网格爬行错步率和圆筒实验趴壁不对称率升高,随后两天稍有好转,但在造模后第4~7天,其神经功能呈进展性恶化,在第7天网格爬行错步率(28.2±0.4%)和圆筒实验趴壁不对称率(24.9±0.6%)均达到峰值,符合临床上进展性缺血性脑卒中患者的神经功能变化特点。
实施例3:小鼠进展性缺血性脑卒中不伴有神经元丢失增加
免疫组织荧光染色:
首先,-20℃预冷的甲醇透化玻片15min。室温下,用1×PBS漂洗2次,每次5min。在室温下用封闭液(PBS+2.5%驴血清/山羊血清,0.2%Triton X-100)封闭1h。在封闭液中稀释特异性一抗(1:200),4℃孵育过夜。次日,于摇床上复温半小时,用1×PBS漂洗3次,每次10min,在黑暗环境中加入相应二抗(1:400)室温孵育1-2h。然后,用1×PBS漂洗3次,每次10min。用含有DAPI的封片液进行封片,盖上盖玻片过程不产生任何气泡,并用透明指甲油密封边缘。4℃避光保存,倒置共聚焦显微镜观察。
神经元死亡是脑卒中神经功能缺损的根本原因,利用免疫组织荧光法观察了缺血后不同时间的小鼠缺血脑组织,结果发现,与假手术组相比,造模1或7天存活神经元(NeuN阳性)均显著下降,但造模1天和造模7天比较无显著差异(图3),提示小鼠进展性缺血性脑卒中不伴有神经元丢失增加。这一病理现象与急性缺血性脑损伤不同,后者通常表现为缺血脑组织半暗带的持续性神经元死亡。可见,本方法构建的小鼠模型为进展性缺血性脑卒中,区别于普通急性脑缺血损伤模型。
实施例4:小鼠进展性缺血性脑卒中伴有迟发性小胶质细胞激活
通过对小胶质细胞标志物Iba-1进行免疫染色观察,结果发现,小胶质细胞在造模后1天无显著变化,但在造模后4天逐渐向损伤区域聚集,造模后4~7天梗死区周边小胶质细胞数量显著增加(图4A-F)。此外,小胶质细胞形态的充实度和坚固性是其激活程度的指标,可见造模后4-7天梗死区周边小胶质细胞呈现持续激活,在造模后7天达到顶峰。小胶质细胞的激活程度与小鼠造模后神经功能的缺损程度在时相上高度一致,这一结果表明,与急性缺血性脑损伤不同,小胶质细胞激活才是进展性卒中发生的关键因素。
Claims (4)
1.一种进展性缺血性脑卒中模型的构建方法,其特征在于所述方法是采用光化学栓塞法,使用的冷光源可见光发光直径为2.0~2.5mm,光源垂直贴于颅骨上方,并使光源圆心较前卤向右偏移1.0~1.5mm;先腹腔注射玫瑰红,剂量为80~100mg/kg,3~5min后进行冷光源照射10~15min,冷光源强度为150000~16000lux,照射结束后后移除冷光源,缝合切口并消毒,腹腔注射青霉素,剂量为2~5万U/kg,将动物置于恒温平板上至其苏醒体温恢复正常,并放回笼内继续饲养14天以上,获得进展性缺血性脑卒中模型。
2.如权利要求1所述的方法,其特征在于所述方法包括如下顺序步骤:
(1)选取10~12周龄、体重23~25g的雄性小鼠,七氟烷气体维持麻醉;
(2)将小鼠头部固定于脑立体定位仪上,剔除头部毛发并表面消毒,通过正中切口剪开皮肤,暴露前囟;
(3)冷光源发光直径为2.0mm,光源垂直贴于颅骨上方,并使光源圆心较前卤向右偏移1.5mm;
(4)先腹腔注射玫瑰红溶液,剂量为100mg/kg,5min后进行冷光源照射15min,冷光源强度为16000lux;
(5)照射结束后后移除冷光源,缝合切口并消毒,腹腔注射青霉素,剂量为5万U/kg;
(6)将动物置于恒温平板上至其苏醒体温恢复正常,并放回笼内继续饲养14天,获得进展性缺血性脑卒中模型。
3.如权利要求2所述的方法,其特征在于所述小鼠为C57BL/6J野生型雄性小鼠。
4.如权利要求2所述的方法,其特征在于步骤(4)中冷光源照射的脑区位于运动皮层。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110769449.4A CN113383750A (zh) | 2021-07-07 | 2021-07-07 | 一种进展性缺血性脑卒中模型的构建方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110769449.4A CN113383750A (zh) | 2021-07-07 | 2021-07-07 | 一种进展性缺血性脑卒中模型的构建方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113383750A true CN113383750A (zh) | 2021-09-14 |
Family
ID=77625414
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110769449.4A Pending CN113383750A (zh) | 2021-07-07 | 2021-07-07 | 一种进展性缺血性脑卒中模型的构建方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113383750A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115067278A (zh) * | 2022-06-17 | 2022-09-20 | 河南中医药大学第一附属医院 | 一种瘀阻脑络证颅内动脉血管延长扩张动脉模型的构建方法 |
-
2021
- 2021-07-07 CN CN202110769449.4A patent/CN113383750A/zh active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115067278A (zh) * | 2022-06-17 | 2022-09-20 | 河南中医药大学第一附属医院 | 一种瘀阻脑络证颅内动脉血管延长扩张动脉模型的构建方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Vidal-Sanz et al. | Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas | |
Salinas-Navarro et al. | Functional and morphological effects of laser-induced ocular hypertension in retinas of adult albino Swiss mice | |
CN106667982B (zh) | 一种制备斑马鱼血栓模型的方法 | |
Grant et al. | Ontogeny of the retina and optic nerve in Xenopus laevis. I. Stages in the early development of the retina | |
Le et al. | Early retinoic acid deprivation in developing zebrafish results in microphthalmia | |
Zeng et al. | The development of eye shape and the origin of lower field myopia in the guinea pig eye | |
CN101979104A (zh) | 多孔壳聚糖支架、神经干细胞多孔壳聚糖支架及其用途 | |
CN109430166A (zh) | 一种学习记忆功能保护机制的研究方法 | |
WO2002089566A1 (fr) | Modele animal de prostatisme interstitiel | |
CN102499135A (zh) | 用于筛选抗血管损伤药物的斑马鱼血管损伤模型及其建立方法和应用 | |
Heun-Johnson et al. | Differential impact of Met receptor gene interaction with early-life stress on neuronal morphology and behavior in mice | |
CN113383750A (zh) | 一种进展性缺血性脑卒中模型的构建方法 | |
CN116686780A (zh) | 一种基于社会应激的ad小鼠抑郁共病模型的构建方法及应用 | |
Aguilar-Arredondo et al. | Memory retrieval-induced activation of adult-born neurons generated in response to damage to the dentate gyrus | |
Easter Jr et al. | Stem cells and regeneration in the retina: What fish have taught us about neurogenesis | |
Yin et al. | In vitro and in vivo methods for studying retinal ganglion cell survival and optic nerve regeneration | |
del Cerro | Retinal transplants | |
Zlomanczuk et al. | Transplanted clonal neural stem-like cells respond to remote photic stimulation following incorporation within the suprachiasmatic nucleus | |
KR102125084B1 (ko) | 신규한 교모세포종 환자 유래 이종 이식 모델 및 이의 용도 | |
Seal et al. | Implementing the chick embryo model to study vestibular developmental disorders | |
Dufour et al. | Prenatal reduction of E14. 5 embryonically fate‐mapped pyramidal neurons in a mouse model of autism | |
Chen et al. | Stereotaxic atlas of the infant rat brain at postnatal days 7–13 | |
CN110215307A (zh) | 光化学诱导局灶性皮质缺血脑卒中模型的建立方法 | |
CN116920126A (zh) | 一种谷氨酸能神经元投射在预治甲基苯丙胺成瘾中的应用 | |
Donate Lagartos et al. | Postnatal Development of Projections of the Postrhinal Cortex to the Entorhinal Cortex in the Rat |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210914 |