JP2008193941A - Cerebrovascular dementia model mouse and production method thereof - Google Patents
Cerebrovascular dementia model mouse and production method thereof Download PDFInfo
- Publication number
- JP2008193941A JP2008193941A JP2007031706A JP2007031706A JP2008193941A JP 2008193941 A JP2008193941 A JP 2008193941A JP 2007031706 A JP2007031706 A JP 2007031706A JP 2007031706 A JP2007031706 A JP 2007031706A JP 2008193941 A JP2008193941 A JP 2008193941A
- Authority
- JP
- Japan
- Prior art keywords
- carotid artery
- common carotid
- cerebral
- model mouse
- blood flow
- 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
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 208000013677 cerebrovascular dementia Diseases 0.000 title abstract description 11
- 210000001168 carotid artery common Anatomy 0.000 claims abstract description 55
- 241001465754 Metazoa Species 0.000 claims abstract description 31
- 206010012289 Dementia Diseases 0.000 claims abstract description 25
- 238000001356 surgical procedure Methods 0.000 claims abstract description 19
- 210000004720 cerebrum Anatomy 0.000 claims abstract description 15
- 238000012360 testing method Methods 0.000 claims abstract description 14
- 230000003902 lesion Effects 0.000 claims abstract description 12
- 230000017531 blood circulation Effects 0.000 claims abstract description 9
- 230000002490 cerebral effect Effects 0.000 claims abstract description 8
- 210000004885 white matter Anatomy 0.000 claims abstract description 8
- 206010048964 Carotid artery occlusion Diseases 0.000 claims description 21
- 230000003447 ipsilateral effect Effects 0.000 claims description 10
- 238000000034 method Methods 0.000 abstract description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 43
- 230000003727 cerebral blood flow Effects 0.000 description 30
- 241000699670 Mus sp. Species 0.000 description 22
- 230000007423 decrease Effects 0.000 description 13
- 230000003920 cognitive function Effects 0.000 description 12
- 210000004556 brain Anatomy 0.000 description 11
- 210000000877 corpus callosum Anatomy 0.000 description 10
- 102000004127 Cytokines Human genes 0.000 description 9
- 108090000695 Cytokines Proteins 0.000 description 9
- 206010072731 White matter lesion Diseases 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 206010002091 Anaesthesia Diseases 0.000 description 7
- 108090001005 Interleukin-6 Proteins 0.000 description 7
- 208000006011 Stroke Diseases 0.000 description 7
- 230000037005 anaesthesia Effects 0.000 description 7
- 230000002146 bilateral effect Effects 0.000 description 7
- 206010008120 Cerebral ischaemia Diseases 0.000 description 6
- 210000004126 nerve fiber Anatomy 0.000 description 6
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 5
- 206010008118 cerebral infarction Diseases 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 229960002725 isoflurane Drugs 0.000 description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 4
- 201000006474 Brain Ischemia Diseases 0.000 description 4
- 108090000174 Interleukin-10 Proteins 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 4
- 230000003542 behavioural effect Effects 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000002980 postoperative effect Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 206010069729 Collateral circulation Diseases 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 210000003050 axon Anatomy 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 238000000879 optical micrograph Methods 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 210000003625 skull Anatomy 0.000 description 3
- LHYQAEFVHIZFLR-UHFFFAOYSA-L 4-(4-diazonio-3-methoxyphenyl)-2-methoxybenzenediazonium;dichloride Chemical compound [Cl-].[Cl-].C1=C([N+]#N)C(OC)=CC(C=2C=C(OC)C([N+]#N)=CC=2)=C1 LHYQAEFVHIZFLR-UHFFFAOYSA-L 0.000 description 2
- 208000031104 Arterial Occlusive disease Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- -1 IL-1β Proteins 0.000 description 2
- 208000031481 Pathologic Constriction Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 206010053648 Vascular occlusion Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 208000021328 arterial occlusion Diseases 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- 210000001653 corpus striatum Anatomy 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000003412 degenerative effect Effects 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000003193 general anesthetic agent Substances 0.000 description 2
- 230000017306 interleukin-6 production Effects 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 210000001577 neostriatum Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- 230000036262 stenosis Effects 0.000 description 2
- 208000037804 stenosis Diseases 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012353 t test Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000016192 Demyelinating disease Diseases 0.000 description 1
- 206010012305 Demyelination Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- LUWJPTVQOMUZLW-UHFFFAOYSA-N Luxol fast blue MBS Chemical compound [Cu++].Cc1ccccc1N\C(N)=N\c1ccccc1C.Cc1ccccc1N\C(N)=N\c1ccccc1C.OS(=O)(=O)c1cccc2c3nc(nc4nc([n-]c5[n-]c(nc6nc(n3)c3ccccc63)c3c(cccc53)S(O)(=O)=O)c3ccccc43)c12 LUWJPTVQOMUZLW-UHFFFAOYSA-N 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 102000008763 Neurofilament Proteins Human genes 0.000 description 1
- 108010088373 Neurofilament Proteins Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 201000004810 Vascular dementia Diseases 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000007844 axonal damage Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000001159 caudate nucleus Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000000971 hippocampal effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000013227 male C57BL/6J mice Methods 0.000 description 1
- 230000007087 memory ability Effects 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 210000003657 middle cerebral artery Anatomy 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 210000002637 putamen Anatomy 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000002739 subcortical effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 210000001186 vagus nerve Anatomy 0.000 description 1
- 208000021331 vascular occlusion disease Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000003936 working memory Effects 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
【課題】手術手技が簡便であって、動物への負担が軽く、効率的に脳血管性認知症モデルマウスを作製する方法、及び大脳白質に病変を有する脳血管性認知症モデルマウス及びその使用方法等を提供する。
【解決手段】片側の総頸動脈が永久閉塞されており、物体認識試験による識別指数が対照動物と比較して有意に低いことを特徴とする、認知症モデルマウス、およびその作成方法。該マウスの術後の大脳半球の血流量は術前の５０％以上であり、大脳白質に病変を有する。
【選択図】なしA method for efficiently producing a cerebrovascular dementia model mouse having a simple surgical technique, a light burden on animals, and a cerebrovascular dementia model mouse having a lesion in the cerebral white matter and use thereof Provide methods etc.
A dementia model mouse characterized in that a common carotid artery on one side is permanently occluded and a discrimination index by an object recognition test is significantly lower than that of a control animal, and a method for producing the same. The blood flow in the cerebral hemisphere after the operation of the mouse is 50% or more before the operation, and there is a lesion in the cerebral white matter.
[Selection figure] None
Description
本発明は、脳血管性認知症モデル動物に関する。より具体的には、本発明は、大脳白質病変を有する認知症モデル動物、その作製方法及びその使用方法等に関する。 The present invention relates to a cerebrovascular dementia model animal. More specifically, the present invention relates to a dementia model animal having a cerebral white matter lesion, a production method thereof, a use method thereof, and the like.
認知症は、変性型認知症及び脳血管性認知症に大別される。欧米ではアルツハイマー病等の変性型認知症が主流であるが、日本では約半数が皮質下血管性認知症等の脳血管性認知症であり、その中でも特に大脳白質の変性を伴うタイプの症例が主である。 Dementia is roughly classified into degenerative dementia and cerebrovascular dementia. In Europe and the United States, degenerative dementia such as Alzheimer's disease is the mainstream, but in Japan about half of the cases are cerebrovascular dementia such as subcortical vascular dementia. The Lord.
脳血管性認知症モデル動物の作製は、主にラットや砂ネズミを用いて行なわれてきた。例えば、ラット又は砂ネズミに対して両側総頸動脈永久閉塞を行なうことにより、慢性の脳虚血又は大脳白質病変が生じ、認知機能の低下又は学習機能の低下が認められることが報告されている（非特許文献１〜４：Tomimoto, H. et al., 2003; Kudo, T. et al., 1990; Wakita, H., et al., 1994; Hattori, H., et al., 1992）。
Cerebrovascular dementia model animals have been mainly produced using rats and sand mice. For example, it has been reported that chronic bilateral common carotid artery permanent occlusion in rats or sand mice causes chronic cerebral ischemia or cerebral white matter lesions, and a decline in cognitive function or learning function is observed. (Non-Patent
マウスは、遺伝子改変動物が利用できる点、成長が早く、多くの個体を容易に扱える点などにおいてこれらの動物よりも有利である。そこで、マウスについても同様の両側総頸動脈永久閉塞システムの導入が試みられたが、マウスは側副血行路の発達が不十分であること等により２４時間以内に死亡してしまい、成功しなかった（非特許文献５，６：Yang, G., et al., 1997; Wellons, JC et al., 2000）。別の研究者によってマイクロコイルを用いるマウスの両側総頸動脈永久狭窄システムが開発され、この方法で慢性の脳虚血及び大脳白質病変を生じさせ得ることが判明した。しかし、この方法は施術に熟練を要するうえ、この方法によっても術後１週間までの死亡率が１３〜１８％と高率であった。また、この両側総頸動脈永久狭窄システムによる認知機能への影響及びこのマウスの認知症モデルとしての利用の可能性は検討されていない(非特許文献７：Shibata et al., 2004)。
Mice are advantageous over these animals in that genetically modified animals can be used, growth is fast, and many individuals can be easily handled. Therefore, an attempt was made to introduce a similar bilateral common carotid artery permanent occlusion system for mice, but the mice died within 24 hours due to insufficient development of collateral circulation, etc., and were not successful. (Non-Patent
従来、認知症モデル作製の目的ではマウスの片側総頸動脈閉塞は試みられていなかった。マウスにおいて片側ではなく両側総頸動脈永久閉塞が行なわれてきた理由の一つは、ラットにおける研究において両側総頸動脈永久閉塞を施しても大脳白質病変の形成まで長期間が必要であることが知られていたことである。すなわち、寿命の短いマウスではより短期間に病変を形成しなければならないが、片側総頸動脈閉塞では両側総頸動脈閉塞よりも病変の形成に時間がかかるため、病変の形成が困難又は不可能であると考えられていた。 Conventionally, unilateral common carotid artery occlusion of mice has not been attempted for the purpose of creating a dementia model. One of the reasons why bilateral common carotid artery permanent occlusion has been performed in mice, rather than unilateral, is that long-term development of cerebral white matter lesions is required even in cases where bilateral common carotid artery permanent occlusion is performed in rats It is known. In other words, in short-lived mice, lesions must be formed in a shorter period of time, but unilateral common carotid artery occlusion takes more time to form than bilateral common carotid artery occlusion, making it difficult or impossible to form lesions. It was thought to be.
マウスに対する片側総頸動脈閉塞は、別の目的で、すなわち脳に病変を生じないように軽度の脳虚血を起こし、側副循環の回復を調べることを目的として行なわれた（非特許文献８：Kitagawa, K., et al., 2005）。この研究においては、いったんマウスに片側総頸動脈閉塞を施した後、同じ側の中大脳動脈を永久閉塞しているが、片側総頸動脈永久閉塞のみ施した場合、脳血流量が４０％以上であれば病変が生じず、ほとんどのマウスで２８日後までの間の脳血流量が約５０％以上であったとされている。また、脳血流量が３５％以下であったマウスは５０匹中６匹で、そのうち１匹に脳梗塞、２匹に海馬の病変が見られたとされている。 Unilateral common carotid artery occlusion for mice was performed for another purpose, that is, for the purpose of examining mild cerebral ischemia so as not to cause lesions in the brain and examining the recovery of collateral circulation (Non-patent Document 8). : Kitagawa, K., et al., 2005). In this study, the mice were once occluded with the common common carotid artery, and then the middle cerebral artery was permanently occluded on the same side. If this is the case, no lesion occurs, and in most mice, the cerebral blood flow was up to about 50% after 28 days. Further, it was reported that 6 out of 50 mice had a cerebral blood flow of 35% or less, of which 1 had cerebral infarction and 2 had hippocampal lesions.
したがって、マウスの片側総頸動脈閉塞によって大脳白質病変又は認知機能もしくは学習機能の低下が起こるとは一般に考えられておらず、また、それが確認された例は報告されていない。 Therefore, it is not generally considered that unilateral common carotid artery occlusion in mice causes cerebral white matter lesions or a decline in cognitive function or learning function, and no examples have been reported.
本発明は、手術手技が簡便であって、動物への負担が軽く、効率的に脳血管性認知症モデルマウスを作製する方法、及び大脳白質に病変を有する脳血管性認知症モデルマウス及びその使用方法等を提供することを目的とする。 The present invention provides a method for efficiently producing a cerebrovascular dementia model mouse with a simple surgical technique, a light burden on animals, and a cerebrovascular dementia model mouse having a lesion in the cerebral white matter and its The purpose is to provide usage.
本発明によれば、
〔１〕 片側の総頸動脈が永久閉塞されており、物体認識試験による識別指数が対照動物と比較して有意に低いことを特徴とする、認知症モデルマウス；
〔２〕 片側総頸動脈閉塞後の同側性大脳半球の血流量が術前の血流量の５０％以上である、前記〔１〕記載の認知症モデルマウス；
〔３〕 大脳白質に病変を有する、請求項１又は２記載の認知症モデルマウス。
〔４〕 マウスの片側の総頸動脈を永久閉塞することを特徴とする、前記〔１〕〜〔３〕のいずれか１項記載の認知症モデルマウスの作製方法、
が提供される。
According to the present invention,
[1] A dementia model mouse characterized in that the common carotid artery on one side is permanently occluded and the discrimination index by the object recognition test is significantly lower than that of a control animal;
[2] The dementia model mouse according to the above [1], wherein the blood flow in the ipsilateral cerebral hemisphere after unilateral common carotid artery occlusion is 50% or more of the blood flow before surgery;
[3] The dementia model mouse according to
[4] The method for producing a dementia model mouse according to any one of [1] to [3], wherein the common carotid artery on one side of the mouse is permanently occluded,
Is provided.
本発明によれば、簡便な手術手技により熟練者でなくても容易に、しかも比較的短期間で脳血管性認知症モデルマウスを作製することができる。また、本発明の方法は動物に対する負担が少なく、術後の死亡率がゼロ又は非常に低いため、効率的に所望のモデルマウスを作製することができる。さらに、マウスを用いているため、種々の遺伝的変異を有する動物を容易に選択して使用することができ、多数の動物を容易に維持・管理できるため、有利である。 According to the present invention, a cerebrovascular dementia model mouse can be easily produced by a simple surgical technique, even if it is not an expert, and in a relatively short period of time. Moreover, since the method of the present invention has less burden on animals and the post-operative mortality rate is zero or very low, a desired model mouse can be efficiently produced. Furthermore, since mice are used, it is advantageous because animals having various genetic mutations can be easily selected and used, and a large number of animals can be easily maintained and managed.
また、本発明のモデルマウスは、大脳白質に病変を有し、脳血管性認知症の主要なタイプの病態を非常によく再現している。さらに、ヒトにおける総頸動脈閉塞は片側に生じるので、その状態をも再現している。したがって、本発明のモデルマウスは、認知症の治療薬又は治療方法の探索、認知症における生理的変化やそれらの変化が認知機能に与える影響の解明などを含む、各種の研究・開発における使用に特に適している。 In addition, the model mouse of the present invention has lesions in the cerebral white matter and reproduces the major types of cerebrovascular dementia very well. Furthermore, since the common carotid artery occlusion in humans occurs on one side, this state is also reproduced. Therefore, the model mouse of the present invention can be used in various research and development, including the search for therapeutic agents or methods for dementia, and the elucidation of the physiological changes in dementia and the effects of those changes on cognitive functions. Especially suitable.
本発明の認知症モデルマウスを作製するための動物は、マウスであればよく、人為的又は自然の遺伝的変異を有している系統であってもよい。認知症症状の発生の点から見て、成体マウス（例えば１２週齢）が好ましく、また、女性ホルモンの一種（エストロゲン）が神経保護作用を持つことが知られているため、雄マウスが好ましい。Ｃ５７Ｂ６／Ｊ系統は、他のマウス系統と比較して側副血行路の発達が悪く（上記非特許文献６参照）、脳虚血に対して脆弱であると考えられるので、特に好ましい。動物の維持・管理については、特に制限はなく、通常の飼育（例えば自由摂食・飲水）を行なうことができる。
The animal for producing the dementia model mouse of the present invention may be a mouse and may be a strain having an artificial or natural genetic variation. From the viewpoint of the occurrence of dementia symptoms, an adult mouse (for example, 12 weeks old) is preferable, and a male mouse is preferable because a female hormone (estrogen) is known to have a neuroprotective action. The C57B6 / J strain is particularly preferable because development of the collateral circulation is poor compared to other mouse strains (see Non-Patent
本発明の方法においては、マウスの片側総頸動脈を永久閉塞する。具体的には、麻酔下でマウスの頸部を切開し、右又は左いずれか一方の総頸動脈を閉塞する。この閉塞は手段を問わないが、一般的には、糸で縛ること（結紮）が特殊な器具を必要とせず、簡便であり、特別な熟練を要しないので、有利である。総頸動脈上の閉塞ヵ所は１ヵ所以上であればよく、２ヵ所〜３ヵ所程度であることが好ましい。最も好ましくは２ヶ所である。これは、総頸動脈の２ヵ所結紮は１ヵ所結紮よりもより高い精度で脳虚血を誘導できる利点が考えられるうえ、２ヵ所結紮は、１ヵ所結紮と比較して手術手技的な負担増は小さいためである。その後、切開部を閉じ、飼育を続ける。なお、ここで、「永久閉塞」とは、一過性の閉塞に対峙する用語であり、閉塞した後に閉塞手段を除く措置を積極的には行なわないことを意味する。 In the method of the present invention, the unilateral common carotid artery of the mouse is permanently occluded. Specifically, the neck of the mouse is incised under anesthesia, and either the right or left common carotid artery is occluded. Any means can be used for this occlusion, but generally, tying with a thread (ligation) is advantageous because it does not require a special instrument, is simple, and does not require special skill. The number of occlusions on the common carotid artery may be one or more, and preferably about 2 to 3 places. Most preferably, there are two locations. This suggests that the ligation of the common carotid artery at two locations can induce cerebral ischemia with higher accuracy than the ligation at one location, and the ligation at two locations increases the surgical technique compared to ligation at one location. Because it is small. Then, the incision is closed and breeding is continued. Here, “permanent occlusion” is a term for temporary occlusion, and means that measures other than occlusion means are not actively performed after occlusion.
本発明の方法によれば、上記のような片側総頸動脈閉塞術後、同側性大脳半球における脳血流量は術前の約６５％程度まで低下し、その後上昇傾向となるが、少なくとも術後２８日までは術前より低い状態を保つことがわかっている。したがって、本発明のモデルマウスは、術後２８日までの任意の時点で、片側総頸動脈閉塞後の同側性大脳半球の脳血流量が術前の脳血流量の４０％以上、好ましくは５０％以上、さらに好ましくは６０％以上である。典型的には、術後２８日までの任意の時点で、片側総頸動脈閉塞後の同側性大脳半球の脳血流量が術前の脳血流量の６５％以上１００％以下（後述の実施例においては６５％以上８８％以下であった）である。 According to the method of the present invention, after unilateral common carotid artery occlusion as described above, the cerebral blood flow in the ipsilateral cerebral hemisphere decreases to about 65% before the operation and then increases, but at least the operation It has been found that it remains lower than before surgery until 28 days later. Therefore, in the model mouse of the present invention, the cerebral blood flow in the ipsilateral cerebral hemisphere after unilateral common carotid artery occlusion is 40% or more of preoperative cerebral blood flow, 50% or more, more preferably 60% or more. Typically, at any time up to 28 days after surgery, the cerebral blood flow in the ipsilateral cerebral hemisphere after unilateral common carotid artery occlusion is 65% or more and 100% or less of preoperative cerebral blood flow (described later). In the example, it was 65% or more and 88% or less).
脳血流量の測定は、術前・術後のマウスについて、麻酔薬イソフルランを１．５％の濃度で用いた麻酔下で、レーザードップラー脳血流計を用いて、頭蓋骨前頂（Bregma）部より後方１mm、正中より側方２．５mmの大脳皮質の血流を、頭蓋骨を通して測定することにより行なうことができる。より正確な測定のためには、同一個体について得た少なくとも１０回、好ましくは２０回以上の測定値を平均してその個体の脳血流量とする。例えば、後述の実施例においては、３０秒間に１．５秒間隔で２１回測定し、その平均値を個体の脳血流量とした。術前に対する術後の相対的な脳血流量（％）は以下の式によって算出する：
脳血流量（％）＝（術後の脳血流量）／（術前の脳血流量）×１００
同様に測定した場合、対側性大脳半球における脳血流量は、術後２８日までの任意の時点で、約６０〜１４０％、より一般的には約８０〜１２０％程度である。典型的には、術後２８日までの任意の時点で、片側総頸動脈閉塞後の対側性大脳半球の脳血流量が術前の脳血流量の９０％以上１２０％以下（後述の実施例においては９２％以上１１６％以下であった）である。
Measurement of cerebral blood flow was performed on pre- and post-operative mice under anesthesia using the anesthetic drug isoflurane at a concentration of 1.5% using a laser Doppler cerebral blood flow meter. The blood flow in the
Cerebral blood flow (%) = (Postoperative cerebral blood flow) / (Preoperative cerebral blood flow) × 100
When measured similarly, the cerebral blood flow in the contralateral cerebral hemisphere is about 60-140%, more typically about 80-120%, at any point up to 28 days after surgery. Typically, at any time up to 28 days after surgery, the cerebral blood flow in the contralateral cerebral hemisphere after unilateral common carotid artery occlusion is 90% or more and 120% or less of preoperative cerebral blood flow (described later). In the example, it was 92% or more and 116% or less).
また、本発明の方法によれば、脳血流量が上記のように保たれているにもかかわらず、片側総頸動脈閉塞術後３０日以内にマウスの大脳白質病変及び／又は認知機能の低下が生じることがわかっている。 In addition, according to the method of the present invention, the cerebral white matter lesion and / or the cognitive function decline in the mouse within 30 days after the unilateral common carotid artery occlusion despite the cerebral blood flow being maintained as described above. Is known to occur.
大脳白質の病変は、一般的な方法によって脳切片試料を作製し、顕微鏡観察することによって定性的に、又は顕微鏡像を画像解析することにより定量的に確認することができる。本発明のモデルマウスは、例えば、術後３０日の時点で、対照群（本明細書においては、特に示さない限り「偽手術群」、すなわち動脈の閉塞を施さないこと以外は同一の手術を行なった群を意味する）と比較して、（１）総頸動脈閉塞側脳梁における神経線維密度の有意な低下、（２）脳梁における軸索数の減少、という特徴の少なくとも一つを呈する。なお、一般に、結紮反対側においては、脳梁、線条体とも神経線維密度の有意な変化は見られない。 The lesion of cerebral white matter can be confirmed qualitatively by preparing a brain slice sample by a general method and observing under a microscope, or quantitatively by analyzing a microscope image. The model mouse of the present invention, for example, at the 30th day after the operation, the control group (in this specification, unless otherwise indicated, the “sham operation group”, that is, the same operation except that the arterial occlusion is not performed) At least one of the following features: (1) a significant decrease in nerve fiber density in the common carotid artery occlusion side corpus callosum, and (2) a decrease in the number of axons in the corpus callosum Present. In general, no significant change in nerve fiber density is observed in the corpus callosum or striatum on the opposite side of ligation.
また、認知機能は、例えば物体認識試験（object recognition test）によって行動評価を行なうことにより確認することができる。この試験は動物が一度記憶した対象物に比べて、新規の対象物に対してより関心を示す性質を利用したもので、前頭葉皮質下回路に関連性のある非空間的作業記憶を評価するものである（Ennaceur A., Delacour J, Behav Brain Res. 1988; 31: 47-59）。具体的には、以下のようにして行なうことができる。 The cognitive function can be confirmed by performing behavioral evaluation by, for example, an object recognition test. This test takes advantage of the nature of interest in new objects compared to objects once memorized by animals and evaluates nonspatial working memory associated with frontal subcortical circuitry. (Ennaceur A., Delacour J, Behav Brain Res. 1988; 31: 47-59). Specifically, it can be performed as follows.
実験装置（例えば縦３０cm、横４５cm、高さ３０cmのガラス箱）及び動物が記憶する複数の対象物（object）を用意する。試験前日、マウスを対象物のない状態で実験装置内を１０分間自由探索させて環境に馴化させておく。試験当日、試行間隔６０分間で以下の２回の試行を行なう。１回目の試行では、２つの同一の対象物を実験装置の両端に置き、マウスを１０分間自由探索させる。２回目の試行では、１回目の試行で用いた対象物の１つを別種の対象物と置き換え、マウスを５分間自由探索させる。各試行において、対象物から１cm以内に鼻を近づけたり、対象物を鼻や髭で触れている状態を探索行動と定義して探索時間を計測する。識別指数は以下のようにして算出する：
識別指数＝Ｎ−Ｆ／Ｎ＋Ｆ
（式中、Ｆは一度記憶した対象物（１回目の試行で用いた対象物と同じ種類の対象物）の探索に費やした時間、Ｎは新しい対象物（１回目の試行と異なる種類の対象物）の探索に費やした時間をそれぞれ表す）
識別指数は、新規対象物の探索により多く割かれた時間の全探索時間に対する割合であり、一度探索した対象物を動物が記憶していれば値が大きくなり、記憶していなければ値が小さくなる。本発明のモデルマウスは、一般に、上記の試験において対照動物（この場合は「無手術群」、すなわち手術を全く行なわない同等の動物群を使用）と比較して有意に識別指数が低い。なお、この場合の有意差検定はｔ検定による。
An experimental apparatus (for example, a glass box having a length of 30 cm, a width of 45 cm, and a height of 30 cm) and a plurality of objects stored by the animal are prepared. The day before the test, the mouse is allowed to acclimatize to the environment by freely searching the experimental apparatus for 10 minutes in the absence of an object. On the day of the test, the following two trials are performed at a trial interval of 60 minutes. In the first trial, two identical objects are placed at both ends of the experimental apparatus and the mouse is allowed to search freely for 10 minutes. In the second trial, one of the objects used in the first trial is replaced with another kind of object, and the mouse is allowed to freely search for 5 minutes. In each trial, a state in which the nose is brought within 1 cm from the object or the object is touched with a nose or a heel is defined as a search action, and the search time is measured. The identification index is calculated as follows:
Discriminating index = NF / N + F
(Where F is the time spent searching for an object once memorized (the same type of object as used in the first trial), N is a new object (an object of a different type from the first trial) Each represents the time spent exploring things)
The identification index is the ratio of the time spent by searching for a new object to the total search time. The value increases if the animal stores the object once searched, and decreases if the object is not stored. Become. The model mice of the present invention generally have a significantly lower discrimination index in the above tests compared to control animals (in this case using the “no surgery group”, ie, an equivalent group of animals without any surgery). In this case, the significant difference test is based on the t test.
本発明のモデルマウスは、一般的に実験動物を用いて行なわれる任意の実験に用いることができる。例えば、新規の認知症治療又は改善薬の候補物質を投与することにより、その効果を判定するために用いることができる。同様に、新規の治療法などが認知症の個体に与える影響について、本発明のモデルマウスを用いて試験することができる。さらに、本発明のモデルマウスそのものをより詳しく調べることにより、認知症における生理的変化（たとえば脳内サイトカイン産生量の変化）や認知機能とそれらの生理的変化との関連性について解明を進めることができる。当業者は、これら以外にも本発明のモデルマウスを用いた各種の有用な研究を容易に企図することができる。 The model mouse of the present invention can be used for any experiment generally performed using experimental animals. For example, it can be used to determine the effect by administering a candidate substance for a novel dementia treatment or ameliorating drug. Similarly, the influence of a novel treatment method or the like on an individual with dementia can be tested using the model mouse of the present invention. Furthermore, by examining the model mouse of the present invention in more detail, it is possible to elucidate the physiological changes in dementia (for example, changes in brain cytokine production) and the relationship between cognitive function and those physiological changes. it can. In addition to these, those skilled in the art can easily contemplate various useful studies using the model mouse of the present invention.
動物の準備
すべての実験は国立長寿医療センター研究所実験動物委員会における「動物実験のためのガイドライン」に従って行った。実験動物は体重２０〜２４ｇの１２週齢オスのＣ５７ＢＬ／６Ｊ系マウス（使用総数６５匹）（日本ＳＬＣ社より購入）を用いた。
Animal Preparation All experiments were performed according to the “Guidelines for Animal Experiments” in the Laboratory for Animal Experiments at National Institute for Longevity Medicine. The experimental animals used were 12-week-old male C57BL / 6J mice (total number of use: 65) weighing 20-24 g (purchased from Japan SLC).
片側総頸動脈閉塞術による認知症モデルマウスの作製
１週間環境に馴化させたマウスは、フェイスマスクを用いて３０％ Ｏ２／７０％ Ｎ２Ｏの混合ガスに、終濃度５％の麻酔薬イソフルランを用いて麻酔導入を行った。吸入麻酔で麻酔導入した後、１．５％のイソフルラン濃度で麻酔状態を維持し、手術を行った（総数６５匹）。頸部を正中線で切開し、右総頸動脈を迷走神経から分離した後、６号の絹糸（ブレード絹製縫合糸、夏目製作所製）を用いて右総頸動脈を二重結紮した。すなわち、約５mmの間隔を空けて右総頸動脈の２ヵ所を結紮した。血管閉塞した後、切開部を縫合・消毒し、すぐにケージに戻した。麻酔薬イソフルランからの覚醒を確認した後、飼育施設に戻した。
Preparation of a dementia model mouse by unilateral common carotid artery occlusion A mouse acclimated to the environment for 1 week was mixed with 30% O 2 /70% N 2 O using a face mask and an anesthetic with a final concentration of 5% Anesthesia was introduced using isoflurane. After induction of anesthesia by inhalation anesthesia, anesthesia was maintained at an isoflurane concentration of 1.5%, and surgery was performed (a total of 65 animals). The neck was incised with a midline, the right common carotid artery was separated from the vagus nerve, and then the right common carotid artery was double ligated using No. 6 silk thread (blade silk suture, manufactured by Natsume Seisakusho). That is, two locations of the right common carotid artery were ligated with an interval of about 5 mm. After vascular occlusion, the incision was sutured and disinfected, and immediately returned to the cage. After confirming the awakening from the anesthetic drug isoflurane, it was returned to the breeding facility.
手術前後とも、すべてのマウスを自由摂食・飲水で飼育した。 Before and after the operation, all mice were bred with free feeding and drinking water.
比較のため、右総頸動脈の結紮を行う以外全く同じ手術手順を行った動物を作製し、対照群（偽手術群）として以下の実験に用いた（総数２０匹）。 For comparison, an animal that was subjected to exactly the same surgical procedure except that the right common carotid artery was ligated was prepared and used as a control group (sham operation group) in the following experiment (20 animals in total).
統計学的解析
すべてのデータは平均±標準偏差で表示した。有意差検定はｔ検定を用いた。ｐ＜０．０５を統計学的に有意であると定義した。
Statistical analysis All data were expressed as mean ± standard deviation. The t-test was used for the significant difference test. p <0.05 was defined as statistically significant.
脳血流の経時的変化の測定
総頸動脈の閉塞側（右側）と反対側（左側）の大脳皮質の血流を測定した。各マウスについて、１．５％イソフルラン麻酔下で、直径２.０mmのレーザードップラー脳血流計プローブ（OmegaFLO-N1、ニューロサイエンス・イデア社製）を用いて、頭蓋骨前頂部より後方１mm、正中より側方２．５mmの頭蓋骨上の脳血流を測定した。あらかじめ手術前のマウスで基準となる脳血流を測定し、結紮後の変化を右総頸動脈結紮後２時間、１日、３日、７日、１４日、２８日後に測定した。脳血流量（ＣＢＦ）は上述のとおり手術前の基準値に対する百分率で表記した。
Measurement of cerebral blood flow over time The blood flow in the cerebral cortex on the occlusion side (right side) and the opposite side (left side) of the common carotid artery was measured. Each mouse was anesthetized with 1.5% isoflurane anesthesia using a 2.0 mm diameter laser Doppler cerebral blood flow probe (OmegaFLO-N1, manufactured by Neuroscience Idea), 1 mm posterior from the front of the skull, from the midline Cerebral blood flow on the lateral 2.5 mm skull was measured. The cerebral blood flow serving as a reference was measured in advance in mice before surgery, and changes after ligation were measured 2 hours, 1 day, 3 days, 7 days, 14 days and 28 days after ligation of the right common carotid artery. The cerebral blood flow (CBF) was expressed as a percentage of the reference value before the operation as described above.
結果を図１に示す。図１において、白四角は対側性（contralateral）、黒丸は同側性（ipsilateral）の大脳半球における血流量（８匹の平均値±標準偏差）を表し、**はｐ＜０．０５、***はｐ＜０．０１を表す。 The results are shown in FIG. In FIG. 1, white squares represent blood flow in the contralateral (i.e. contralateral), black circles represent ipsilateral hemisphere (mean value of 8 animals ± standard deviation), ** represents p <0.05, *** represents p <0.01.
片側総頸動脈閉塞後の平均脳血流は動脈閉塞側（同側性）大脳半球において基準値と比較して有意な低下が認められた。結紮２時間後では脳血流は手術前の約６５．５±１０．３％（平均±標準偏差）にまで低下した。これが最も低下の大きかった時点であり、その後それ以下になることはなかった。結紮３日後、脳血流は回復し始めたが、結紮２８日後まで有意に低値を示した。一方で、動脈閉塞と反対側の（対側性）大脳半球における脳血流は手術前の約９２．０％〜１１５．６％の間で推移したが、手術前と比較して有意な変化ではなかった。 The mean cerebral blood flow after unilateral common carotid artery occlusion was significantly decreased in the arterial occlusion side (ipsilateral) cerebral hemisphere compared to the reference value. Two hours after ligation, cerebral blood flow decreased to about 65.5 ± 10.3% (mean ± standard deviation) before surgery. This was the time when the decline was the greatest, and it never fell below that. After 3 days of ligation, cerebral blood flow began to recover, but remained significantly low until 28 days after ligation. On the other hand, cerebral blood flow in the hemisphere opposite to the artery occlusion (contralateral) changed between about 92.0% to 115.6% before the operation, but a significant change compared to before the operation. It wasn't.
本実験において、対照群（総数２０匹）及び片側総頸動脈結紮（閉塞）群（総数４５匹）のいずれにおいても死亡例はなかった。すなわち、対照群、片側総頸動脈結紮群のいずれにおいても死亡率は０％であった。 In this experiment, there were no deaths in either the control group (total 20 animals) or the unilateral common carotid artery ligation (occlusion) group (total 45 animals). That is, the mortality rate was 0% in both the control group and the unilateral common carotid artery ligation group.
白質病変及びグリア細胞の活性化の組織学的解析
右総頸動脈結紮３０日後、マウスをジエチル・エーテルで深麻酔した後、０．０１Mリン酸緩衝生理食塩水で経心臓的に灌流し、その後、４％（W/V）パラフォルムアルデヒド／０．１Mリン酸バッファーを含む固定液で灌流した。線条体を含む脳ブロックをパラフィン包埋した。ミクロトーム（SM2000R、LEICA社製）を用いて２μm厚のパラフィン切片を作製した。大脳白質の病理変化（脱髄）はルクソール・ファーストブルー染色した切片の線維密度により評価した。具体的には、尾状核被殻の横断線維束及び脳梁の写真画像をデジタルカメラ（Fujix HC-2500、FUJIFILM社製）に接続した顕微鏡を用いて４０倍対物レンズで撮影した（４００倍）。これらの写真画像を、「NIH image」画像処理・解析ソフトウェアを用いて解析した。
Histological analysis of white matter lesions and activation of glial cells 30 days after ligation of the right common carotid artery, mice were deeply anesthetized with diethyl ether, perfused transcardially with 0.01 M phosphate buffered saline, and then Perfusion was performed with a fixative containing 4% (W / V) paraformaldehyde / 0.1 M phosphate buffer. The brain block containing the striatum was embedded in paraffin. Paraffin sections with a thickness of 2 μm were prepared using a microtome (SM2000R, manufactured by LEICA). The pathological change (demyelination) of the cerebral white matter was evaluated by the fiber density of the sections stained with Luxol Fast Blue. Specifically, photographic images of transverse fiber bundles and corpus callosum of caudate nucleus putamen were photographed with a 40 × objective lens using a microscope connected to a digital camera (Fujix HC-2500, manufactured by FUJIFILM) (400 ×). ). These photographic images were analyzed using “NIH image” image processing and analysis software.
軸索損傷は、抗汎神経細糸（pan-neuronal neurofilament）抗体を用いて免疫細胞化学的に評価した。前記の２μm厚のパラフィン切片を、脱パラフィンした後、抗汎神経細糸抗体（ＳＭＩ３１１, Convance; １：５００希釈）を用いて、一晩反応させた。その後、ビオチン化した抗マウスＩｇＧ（Vector Laboratories; １：２００希釈）を用いて1時間反応させ、続いてアビジン−ビオチン ペルオキシダーゼ試薬（Vector Laboratories; １：１００希釈）を用いて１時間反応させた。最後に、免疫反応産物を、ジアミノベンジジン（Vector Laboratories）を用いて可視化した。 Axonal damage was assessed immunocytochemically using an anti-pan-neuronal neurofilament antibody. The 2 μm-thick paraffin section was deparaffinized and then reacted overnight using an anti-pannergic fiber antibody (SMI311, Convance; 1: 500 dilution). Thereafter, reaction was carried out for 1 hour using biotinylated anti-mouse IgG (Vector Laboratories; 1: 200 dilution), followed by reaction for 1 hour using avidin-biotin peroxidase reagent (Vector Laboratories; 1: 100 dilution). Finally, the immune reaction products were visualized using diaminobenzidine (Vector Laboratories).
結果を図２に示す。パネル（Ａ）はルクソール・ファーストブルー染色した閉塞側の光学顕微鏡像の写真、パネル（Ｂ）は解析ソフトによって定量化されたその神経線維密度（即ちルクソール・ファーストブルー染色陽性面積／全面積(％)）を表し、**はｐ＜０．０５を表す。パネル（Ｃ）は、ＳＭＩ３１１抗体で染色した閉塞側脳梁の光学顕微鏡像の写真である。 The results are shown in FIG. Panel (A) is a photograph of a light microscope image of the occluded side stained with Luxor Fast Blue, and Panel (B) is the nerve fiber density quantified by analysis software (ie, Luxor Fast Blue stained positive area / total area (%) )), And ** represents p <0.05. Panel (C) is a photograph of an optical microscope image of the obstructed corpus callosum stained with SMI311 antibody.
結紮３０日後、片側総頸動脈結紮群では、対照群と比較し、閉塞側の脳梁内側部における髄鞘染色の低下（神経線維密度の有意な低下）が認められた。しかし、線条体では変化は認められなかった。反対側の大脳半球においては、脳梁、線条体とも神経線維密度の有意な変化はなく、白質病変は検出されなかった。 30 days after ligation, the unilateral common carotid artery ligation group showed a decrease in myelin staining (significant decrease in nerve fiber density) in the inner side of the corpus callosum on the occluded side compared with the control group. However, no change was observed in the striatum. In the opposite cerebral hemisphere, there was no significant change in nerve fiber density in both the corpus callosum and striatum, and no white matter lesions were detected.
さらに、片側総頸動脈結紮群では脳梁における軸索数の減少が認められた。すなわち、片側総頸動脈永久閉塞を施したマウスでは、総頸動脈閉塞側脳梁内側部におけるＳＭＩ３１１陽性軸索数の低下が認められた。 Furthermore, in the unilateral common carotid artery ligation group, a decrease in the number of axons in the corpus callosum was observed. That is, in mice subjected to unilateral common carotid artery permanent occlusion, a decrease in the number of SMI311 positive axons in the inner side of the common carotid artery occlusion side corpus callosum was observed.
行動評価
片側総頸動脈結紮群及び対照群のマウスについて、術後３０日目に、物体認識試験により行動評価を行った。この実験においては対照群として無手術群を用いた。
Behavioral Evaluation Behavioral evaluation was performed by object recognition test on the unilateral common carotid artery ligation group and the control group of mice on the 30th day after the operation. In this experiment, the non-operative group was used as a control group.
実験装置としては縦３０cm、横４５cm、高さ３０cmのガラス箱（水槽）を用いた。動物が記憶する対象物としてはプラスチック製の高さ６cmの赤色立方体、緑色角錐、青色円柱を用いた。 As an experimental apparatus, a glass box (water tank) having a length of 30 cm, a width of 45 cm, and a height of 30 cm was used. As objects to be memorized by animals, a plastic 6 cm high red cube, green pyramid, and blue cylinder were used.
環境に馴化させるため、試験前日、マウスを対象物のない状態でガラス箱を１０分間自由探索させた。試験当日は試行間隔６０分間の２回の試行を行なった。１回目の試行では、２つの同一の対象物をガラス箱の両端に置き、マウスを１０分間自由探索させた。対象物から１cm以内に鼻を近づけたり、対象物を鼻や髭で触れている状態を探索行動と定義して探索時間を計測した。２回目の試行では、１回目の試行で用いた対象物の１つを別種の対象物と置き換え、マウスを５分間自由探索させた。一度記憶した対象物（１回目の試行で用いた対象物と同じ種類の対象物）の探索に費やした時間（Ｆ）、新しい対象物（１回目の試行と異なる種類の対象物）の探索に費やした時間（Ｎ）をそれぞれ計測した。記憶機能を評価する指数である識別指数（Ｎ−Ｆ／Ｎ＋Ｆ）の値を算出し、群間で比較した。 In order to acclimatize to the environment, the mouse was allowed to freely search the glass box for 10 minutes without the object the day before the test. On the test day, two trials with a trial interval of 60 minutes were performed. In the first trial, two identical objects were placed at both ends of the glass box and the mouse was allowed to freely explore for 10 minutes. The search time was measured by defining the state where the nose was brought within 1 cm from the object or the object was touched with the nose or the heel as the search action. In the second trial, one of the objects used in the first trial was replaced with another kind of object, and the mouse was allowed to freely search for 5 minutes. For searching for a new object (an object of a different type from the first trial), time (F) spent searching for an object once memorized (an object of the same type as the object used in the first trial) Each time (N) spent was measured. The value of the discrimination index (NF / N + F), which is an index for evaluating the memory function, was calculated and compared between the groups.
なお、本実験では、各試行間で対象物の位置や役割（１回目の試行に用いるか、２回目の試行に用いるか）を無作為的に変更することや対象物、ガラス箱を７０％エタノールで十分に洗浄することによって、場所嗜好性や嗅覚刺激による実験結果への影響を排除した。 In this experiment, the position and role of an object (whether it is used for the first trial or the second trial) is randomly changed between trials, or 70% of the object and the glass box are used. Thorough washing with ethanol eliminated the influence of place preference and olfactory stimuli on the experimental results.
結果を図３に示す。１回目の試行において、片側総頸動脈結紮群と対照群とで識別指数の有意な変化は認められなかった。２回目の試行において、対照群は見慣れた物体よりも新しい物体を探索することに多くの時間を費やした。すなわち、手術後の識別指数では、対照群は対象物を記憶していたが、片側総頸動脈閉塞群では、記憶能力が有意に低下していた。なお、手術前の試行において、片側総頸動脈閉塞群、対照群間で識別指数の有意な差はなく、両者とも対象物を記憶していることが確認された。 The results are shown in FIG. In the first trial, there was no significant change in the discrimination index between the unilateral common carotid artery ligation group and the control group. In the second trial, the control group spent more time searching for new objects than familiar objects. That is, in the discrimination index after surgery, the control group memorized the object, but the memory ability was significantly reduced in the unilateral common carotid artery occlusion group. In the trial before surgery, there was no significant difference in discrimination index between the unilateral common carotid artery occlusion group and the control group, and it was confirmed that both memorized the object.
従来、認知症モデルラットについては、術後６０日〜９０日での認知機能が検討されていた。片側総頸動脈閉塞を施した本発明のマウスは、遅くとも術後３０日という従来と比較して極めて早い時点で既に認知症の症状を呈することが確認された。
Conventionally, for dementia model rats, the
サイトカインの免疫学的測定
特異性の高い抗体及び高感度のＥＬＩＳＡ法を用いて、右総頸動脈結紮後の閉塞側脳（大脳右半球）における炎症性及び抗炎症性サイトカインの産生を定量化した。
Immunoassay for cytokines Highly specific antibodies and sensitive ELISA were used to quantify the production of inflammatory and anti-inflammatory cytokines in the occluded brain (right hemisphere) after ligation of the right common carotid artery .
結紮後２時間、１日後、３日後、７日後、２ヵ月後、及び偽手術群のマウスからサンプル脳を得た。各マウスから全脳を大脳半球ごとに回収し、１mlの２％（w/v）ＳＤＳ含有ＴＢＳ（１０mM Ｔｒｉｓ−ＨＣｌ（pH８．０）、１５０mM ＮａＣｌ）で均質化した後、１００，０００×ｇ（重力）、１時間、４℃の条件で遠心分離した（BECKMAN社、Optima（登録商標）TLX Ultracentrifuge）。得られた上清（可溶性画分）中のＴＮＦ−α、ＩＬ−１β、ＩＬ−６、ＩＬ−４、ＩＬ−１０濃度を、マウスＥＬＩＳＡキット（ENDOGEN社製）を用いて高感度ＥＬＩＳＡ法で測定した。
Sample brains were obtained from
結果を図４に示す。図４は、片側総頸動脈結紮後の脳内の炎症性サイトカイン（ＴＮＦ−α（パネルＡ）、ＩＬ−１β（パネルＢ）、ＩＬ−６（パネルＣ））及び抗炎症性サイトカイン（ＩＬ−４（パネルＤ）、ＩＬ−１０（パネルＥ））濃度をそれぞれ表す。また、Ｓ：偽手術群、２Ｈ：片側総頸動脈結紮後２時間でサンプリング、Ｄ１：同１日でサンプリング、Ｄ３：同３日でサンプリング、Ｄ７：同７日でサンプリング、Ｍ２：同２ヵ月でサンプリングしたサンプルをそれぞれ表す。急性期（結紮３日以内）では、閉塞群は対照群と比較して、総頸動脈閉塞側大脳半球の炎症性サイトカイン（ＴＮＦ−α、ＩＬ−１β、ＩＬ−６）の有意な産生亢進と抗炎症性サイトカイン（ＩＬ−４、ＩＬ−１０）の有意な産生低下が確認された。その後、全てのサイトカイン濃度の変化は７日以内に基準値に戻った。しかし、ＩＬ−６のみが再び慢性期（７日以降）に閉塞群において有意に産生が亢進した。
The results are shown in FIG. FIG. 4 shows inflammatory cytokines (TNF-α (panel A), IL-1β (panel B), IL-6 (panel C)) and anti-inflammatory cytokines (IL−) after unilateral common carotid artery ligation. 4 (panel D) and IL-10 (panel E)) concentrations. In addition, S: sham operation group, 2H: sampling 2 hours after unilateral common carotid artery ligation, D1: sampling on the same day, D3: sampling on the
これまでに、中枢神経系の疾患患者でＩＬ−６産生が亢進すること（Benveniste EN, Cytokine Growth Factor Rev. 1998;9:259-75; Gruol DL and Nelson TE., Mol Neurobiol. 1997;15:307-39）や末梢血中のＩＬ−６産生量と認知機能との間に負の相関があることが報告されている（Weight CB et al., J Stroke Cerebrovasc Dis. 2006;15:34-38）が、サイトカインの産生亢進が認知機能にどのように影響するかについては分かっていない。本発明のモデルマウスは、脳虚血後慢性期にＩＬ−６の産生亢進が確認されたことから、ＩＬ−６と認知機能との関連性について検討できる研究ツールとしても有用であることが示された。 To date, IL-6 production is increased in patients with diseases of the central nervous system (Benveniste EN, Cytokine Growth Factor Rev. 1998; 9: 259-75; Gruol DL and Nelson TE., Mol Neurobiol. 1997; 15: 307-39) and a negative correlation has been reported between IL-6 production in peripheral blood and cognitive function (Weight CB et al., J Stroke Cerebrovasc Dis. 2006; 15: 34- 38) However, it is not known how increased production of cytokines affects cognitive function. Since the increased production of IL-6 was confirmed in the chronic phase after cerebral ischemia, the model mouse of the present invention was also useful as a research tool for examining the relationship between IL-6 and cognitive function. It was done.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007031706A JP2008193941A (en) | 2007-02-13 | 2007-02-13 | Cerebrovascular dementia model mouse and production method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007031706A JP2008193941A (en) | 2007-02-13 | 2007-02-13 | Cerebrovascular dementia model mouse and production method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2008193941A true JP2008193941A (en) | 2008-08-28 |
Family
ID=39753484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2007031706A Pending JP2008193941A (en) | 2007-02-13 | 2007-02-13 | Cerebrovascular dementia model mouse and production method thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2008193941A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010087306A1 (en) | 2009-01-29 | 2010-08-05 | 株式会社林原生物化学研究所 | Anti-neurodegenerative disease agent |
| CN102512259A (en) * | 2011-11-18 | 2012-06-27 | 新乡医学院 | Novel method for establishing vascular dementia rat model |
| WO2022035049A1 (en) * | 2020-08-13 | 2022-02-17 | 동국대학교 산학협력단 | Novel method for preparing animal model of cerebrovascular disease and method for producing animal having small individual difference in susceptibility to cerebrovascular disease onset by using animal model for cerebrovascular disease prepared thereby |
| CN118696874A (en) * | 2024-05-28 | 2024-09-27 | 徐阳 | A method for constructing an animal model of chronic cognitive impairment |
-
2007
- 2007-02-13 JP JP2007031706A patent/JP2008193941A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| JPN6012027673; Ｓｔｒｏｋｅ Ｖｏｌ．３６， Ｎｏ．１０, 200510, ｐｐ．２２７０-２２７４ * |
| JPN6012027675; Ａｎｎｕａｌ Ｒｅｖｉｅｗ ｏｆ Ｐｈａｒｍａｃｏｌｏｇｙ ａｎｄ Ｔｏｘｉｃｏｌｏｇｙ Ｖｏｌ．２９, 1989, ｐｐ．４０３-４１４ * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010087306A1 (en) | 2009-01-29 | 2010-08-05 | 株式会社林原生物化学研究所 | Anti-neurodegenerative disease agent |
| CN102512259A (en) * | 2011-11-18 | 2012-06-27 | 新乡医学院 | Novel method for establishing vascular dementia rat model |
| WO2022035049A1 (en) * | 2020-08-13 | 2022-02-17 | 동국대학교 산학협력단 | Novel method for preparing animal model of cerebrovascular disease and method for producing animal having small individual difference in susceptibility to cerebrovascular disease onset by using animal model for cerebrovascular disease prepared thereby |
| KR20220021933A (en) * | 2020-08-13 | 2022-02-23 | 동국대학교 산학협력단 | Methods for preparing a novel cerebrovascular disease animal model and for the model-based selective breeding to generate offsprings having a similar susceptibility to cerebrovasculr diseases |
| KR102545491B1 (en) | 2020-08-13 | 2023-06-21 | 동국대학교 산학협력단 | Methods for preparing a novel cerebrovascular disease animal model and for the model-based selective breeding to generate offsprings having a similar susceptibility to cerebrovasculr diseases |
| CN118696874A (en) * | 2024-05-28 | 2024-09-27 | 徐阳 | A method for constructing an animal model of chronic cognitive impairment |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Vuillermot et al. | A longitudinal examination of the neurodevelopmental impact of prenatal immune activation in mice reveals primary defects in dopaminergic development relevant to schizophrenia | |
| West et al. | Binge ethanol effects on prefrontal cortex neurons, spatial working memory and task-induced neuronal activation in male and female rats | |
| Freeman et al. | Transplanted fetal striatum in Huntington's disease: phenotypic development and lack of pathology | |
| Antonow‐Schlorke et al. | Glucocorticoid exposure at the dose used clinically alters cytoskeletal proteins and presynaptic terminals in the fetal baboon brain | |
| Yuan et al. | Abnormal frontostriatal tracts in young male tobacco smokers | |
| Goldberg et al. | Social enrichment attenuates nigrostriatal lesioning and reverses motor impairment in a progressive 1-methyl-2-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease | |
| Chen et al. | Chronic exposure to hypoxia inhibits myelinogenesis and causes motor coordination deficits in adult mice | |
| Ryding et al. | Regional brain serotonin and dopamine transporter binding capacity in suicide attempters relate to impulsiveness and mental energy | |
| Lopes et al. | Glymphatic inhibition exacerbates tau propagation in an Alzheimer’s disease model | |
| Testen et al. | Region-specific differences in morphometric features and synaptic colocalization of astrocytes during development | |
| Sarubbo et al. | Uncovering the inferior fronto-occipital fascicle and its topological organization in non-human primates: the missing connection for language evolution | |
| Vafaei-Nezhad et al. | The impacts of diabetes in pregnancy on hippocampal synaptogenesis in rat neonates | |
| US20240398856A1 (en) | Methods of Modifying Neuronal Function by Changing Intracellular Magnesium Levels | |
| Grubb et al. | Biochemical and anatomical subdivision of the dorsal lateral geniculate nucleus in normal mice and in mice lacking the β2 subunit of the nicotinic acetylcholine receptor | |
| JP2008193941A (en) | Cerebrovascular dementia model mouse and production method thereof | |
| Seelke et al. | Differential changes in the cellular composition of the developing marsupial brain | |
| Lee et al. | Carvacrol inhibits expression of transient receptor potential melastatin 7 channels and alleviates zinc neurotoxicity induced by traumatic brain injury | |
| López‐Cervantes et al. | Cerebellar spongiform degeneration is accompanied by metabolic, cellular, and motor disruption in male rats with portacaval anastomosis | |
| Munger et al. | Comparative analysis of astrocytes in the prefrontal cortex of primates: Insights into the evolution of human brain energetics | |
| Guo et al. | Mechanism of Gastrodin against neurotoxicity based on network pharmacology, molecular docking and experimental verification | |
| Triarhou et al. | Stabilisation of neurone number in the inferior olivary complex of aged ‘Purkinje cell degeneration’mutant mice | |
| Lv et al. | Abdominal surgery plus sevoflurane exposure induces abnormal emotional changes and cognitive dysfunction in aged rats | |
| Ferguson et al. | Impact of gulf war toxic exposures after mild traumatic brain injury | |
| Odaci et al. | Effects of low‐dose oxcarbazepine administration on developing cerebellum in newborn rat: A stereological study | |
| Yoshino et al. | Long-term impact and clinical significance of living donor liver transplantation with respect to donor liver restoration and spleen size: a prospective study |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Effective date: 20100129 Free format text: JAPANESE INTERMEDIATE CODE: A621 |
|
| A131 | Notification of reasons for refusal |
Effective date: 20120605 Free format text: JAPANESE INTERMEDIATE CODE: A131 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20121016 |