著者は、批判的に原稿を編集して再検討し、証明にジェイデン·スミスに感謝します。この作品は、国立多発性硬化症協会（NMSS、部分的な補助金RG-4001-A1）から支持を受けて、イタリアの多発性硬化症協会（AISMは、2010/R/31を付与）、イタリア保健省（GR08-7）、ライフ、バンカアグリコラPOPOLAREディラグーザ（BAPR）、欧州研​​究会議（ERC）ERC-2010-STGグラント契約の下では260511-SEM_SEMと欧州共同体（EC）第7次フレームワークプログラム（FP7/2007-2013）のための翼グラント契約書N *度の下で; 280772 - ジオン。

<ol>
	<li>Ben-Hur, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+multipotential+neural+precursor+cells+migrate+into+the+inflamed+white+matter+in+response+to+experimental+autoimmune+encephalomyelitis.">Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis.</a> Glia. 41, 73-80 (2003).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+adult+neurospheres+induces+recovery+in+a+chronic+model+of+multiple+sclerosis.">Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis.</a> Nature. 422, 688-694 (2003).</li><li>Chu, K., Kim, M., Jeong, S. W., Kim, S. U., Yoon, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+can+migrate,+differentiate,+and+integrate+after+intravenous+transplantation+in+adult+rats+with+transient+forebrain+ischemia.">Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia.</a> Neurosci. Lett. 343, 129-133 (2003).</li><li>Bottai, D., Madaschi, L., Di Giulio, A. M., Gorio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability-dependent+promoting+action+of+adult+neural+precursors+in+spinal+cord+injury.">Viability-dependent promoting action of adult neural precursors in spinal cord injury.</a> Mol. Med. 14, 634-644 (2008).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere-derived+multipotent+precursors+promote+neuroprotection+by+an+immunomodulatory+mechanism.">Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism.</a> Nature. 436, 266-271 (2005).</li><li>Einstein, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraventricular+transplantation+of+neural+precursor+cell+spheres+attenuates+acute+experimental+allergic+encephalomyelitis.">Intraventricular transplantation of neural precursor cell spheres attenuates acute experimental allergic encephalomyelitis.</a> Mol. Cell Neurosci. 24, 1074-1082 (2003).</li><li>Chu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cell+transplantation+reduces+spontaneous+recurrent+seizures+following+pilocarpine-induced+status+epilepticus+in+adult+rats.">Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats.</a> Brain Res. 1023, 213-221 (2004).</li><li>Jeong, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cell+transplantation+promotes+functional+recovery+in+rats+with+experimental+intracerebral+hemorrhage.">Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage.</a> Stroke. 34, 2258-2263 (2003).</li><li>Aboody, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+display+extensive+tropism+for+pathology+in+adult+brain:+evidence+from+intracranial+gliomas.">Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 12846-12851 (2000).</li><li>Muller, F. J., Snyder, E. Y., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy:+can+neural+stem+cells+deliver.">Gene therapy: can neural stem cells deliver.</a> Nat. Rev. Neurosci. 7, 75-84 (2006).</li><li>Martino, G., Pluchino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+potential+of+neural+stem+cells.">The therapeutic potential of neural stem cells.</a> Nat. Rev. Neurosci. 7, 395-406 (2006).</li><li>Deckert-Schluter, M., Schluter, D., Hof, H., Wiestler, O. D., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+ICAM-1,+VCAM-1+and+their+ligands+LFA-1,+Mac-1,+CD43,+VLA-4,+and+MHC+class+II+antigens+in+murine+Toxoplasma+encephalitis:+a+light+microscopic+and+ultrastructural+immunohistochemical+study.">Differential expression of ICAM-1, VCAM-1 and their ligands LFA-1, Mac-1, CD43, VLA-4, and MHC class II antigens in murine Toxoplasma encephalitis: a light microscopic and ultrastructural immunohistochemical study.</a> J. Neuropathol. Exp. Neurol. 53, 457-468 (1994).</li><li>Hemmer, B., Archelos, J. J., Hartung, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+the+immunopathogenesis+of+multiple+sclerosis.">New concepts in the immunopathogenesis of multiple sclerosis.</a> Nat. Rev. Neurosci. 3, 291-301 (2002).</li><li>Butcher, E. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+homing+and+homeostasis.">Lymphocyte homing and homeostasis.</a> Science. 272, 60-66 (1996).</li><li>Rampon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+systemic+delivery+of+neural+precursor+cells+to+the+brain:+assembly+of+brain+endothelial+apical+cups+and+control+of+transmigration+by+CD44.">Molecular mechanism of systemic delivery of neural precursor cells to the brain: assembly of brain endothelial apical cups and control of transmigration by CD44.</a> Stem Cells. 26, 1673-1682 (2008).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+ameliorate+autoimmune+encephalomyelitis+in+non-human+primates.">Human neural stem cells ameliorate autoimmune encephalomyelitis in non-human primates.</a> Ann. Neurol. 66, 343-354 (2009).</li><li>Martino, G., Pluchino, S., Bonfanti, L., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+regeneration+in+physiology+and+pathology:+the+immune+signature+driving+therapeutic+plasticity+of+neural+stem+cells.">Brain regeneration in physiology and pathology: the immune signature driving therapeutic plasticity of neural stem cells.</a> Physiol. Rev. 91, 1281-1304 (2011).</li><li>Aharonowiz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+transplanted+human+embryonic+stem+cell-derived+neural+precursors+in+an+animal+model+of+multiple+sclerosis.">Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis.</a> PLoS ONE. 3, e3145 (2008).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulatory+neural+stem/precursor+cells+protect+from+central+nervous+system+autoimmunity+by+restraining+dendritic+cell+function.">Immune regulatory neural stem/precursor cells protect from central nervous system autoimmunity by restraining dendritic cell function.</a> PLoS One. 4, (2009).</li><li>Einstein, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+attenuate+autoimmune+encephalomyelitis+by+peripheral+immunosuppression.">Neural precursors attenuate autoimmune encephalomyelitis by peripheral immunosuppression.</a> Ann. Neurol. 61, 209-218 (2007).</li><li>Gritti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotential+stem+cells+from+the+adult+mouse+brain+proliferate+and+self-renew+in+response+to+basic+fibroblast+growth+factor.">Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.</a> J. Neurosci. 16, 1091-1100 (1996).</li><li>Furlan, R., Pluchino, S., Marconi, P. C., Martino, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+gene+delivery+into+the+central+nervous+system+using+intrathecally+injected+nonreplicative+viral+vectors.">Cytokine gene delivery into the central nervous system using intrathecally injected nonreplicative viral vectors.</a> Methods Mol. Biol. 215, 279-289 (2003).</li><li>Constantin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+analysis+of+adhesive+events+in+brain+microvessels+by+using+intravital+microscopy.">Visualization and analysis of adhesive events in brain microvessels by using intravital microscopy.</a> Methods Mol. Biol. 239, 189-198 (2004).</li><li>Politi, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic-resonance-based+tracking+and+quantification+of+intravenously+injected+neural+stem+cell+accumulation+in+the+brains+of+mice+with+experimental+multiple+sclerosis.">Magnetic-resonance-based tracking and quantification of intravenously injected neural stem cell accumulation in the brains of mice with experimental multiple sclerosis.</a> Stem Cells. 25, 2583-2592 (2007).</li><li>Melzi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-graft+of+allogeneic+immune+regulatory+neural+stem+cells+(NPC)+and+pancreatic+islets+mediates+tolerance,+while+inducing+NPC-derived+tumors+in+mice.">Co-graft of allogeneic immune regulatory neural stem cells (NPC) and pancreatic islets mediates tolerance, while inducing NPC-derived tumors in mice.</a> PLoS One. 5, (2010).</li><li>Amariglio, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-derived+brain+tumor+following+neural+stem+cell+transplantation+in+an+ataxia+telangiectasia+patient.">Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient.</a> PLoS Med. 6, (2009).</li><li>Ben-Hur, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+proinflammatory+cytokines+on+the+growth,+fate,+and+motility+of+multipotential+neural+precursor+cells.">Effects of proinflammatory cytokines on the growth, fate, and motility of multipotential neural precursor cells.</a> Mol. Cell Neurosci. 24, 623-631 (2003).</li><li>Giusto, E., Donega, M., Cossetti, C., Pluchino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuro-immune+interactions+of+neural+stem+cell+transplants:+From+animal+disease+models+to+human+trials.">Neuro-immune interactions of neural stem cell transplants: From animal disease models to human trials.</a> Exp. Neurol. , (2013).</li></ol>

著者らは、開示することは何もありません。

体性幹細胞に基づく治療は、例えば、MS2 11などの慢性炎症性CNS障害を治療するための最も有望な戦略の一つとして浮上している。その治療効果を維持するメカニズムはまだ完全に解明される必要があるが、神経変性疾患の様々な実験モデルにおいて、NPC移植の重大な影響は、細胞がすぐにヒトでの研究にも適用することができる幹やや挑発的な信念に上昇を与えている。しかし、我?...

強力な証拠は、CNS障害1-8の動物モデルにおける体性神経幹/前駆細胞（NPCの）移植の治療効果を証明するインビボ研究から生じた。これらの実験結果は臨床応用に変換することができる前に、それにもかかわらず、宿主への幹細胞の送達に関連する問題の数は、慎重な検討が必要です。多病巣性、慢性炎症性脳疾患のための（非造血系）回復幹細胞治療の開発に向け、特に、実質的なハードルは、NPCの注射の理想的なルートの同定である。対象となる疾患の病態生理をしっかり理解（限局性または多病巣性、原発性または原発性変性）、および配信技術に関連する実現可能性とリスクの問題の慎重な分析は、幹細胞送達に最適なプロトコルを識別している。 （焦点間<em&gt;例えば。神経系の実質に）幹細胞移植損傷の空間的に閉じ込められた領域によって特徴付けられるCNS疾患の治療への論理的アプローチである（ 例えば、パーキンソン病およびハンチントン病、脳および脊髄外傷、および脳卒中）、非常に同じアプローチを証明することができるこのような多病巣性、慢性、および空間的に播種性CNS損傷が時間をかけて蓄積し、MS、などの条件に実質的に実行不可能であると。この後者の場合、個々の病変に焦点細胞注入を標的はまた、このように低侵襲NPCの移植に標的CNSの他の、より適切な方法の識別を促す、CNS実質内長距離にわたって移行する移植されたNPCの限られた能力によって妨げられる。 大きな期待はCNS9外に血管内に注入された場合のNPCは、マウスにおける頭蓋内腫瘍（ 例えば神経膠腫）を標的観測から明らかになった。この独創的な次の幹細胞pathotrophism 10のin vivoでの証拠に 、広範なデータは、静脈内のいずれかを介して 、炎症性CNS損傷のモデルとして、実験的自己免疫性脳脊髄炎（EAE）と実験動物における実現可能性とのNPCの全身移植の治療効果に関する蓄積されてきた（IV）または脳室内（ICV）のNPC注入1,2,5,6,8···私たちは、まず、これはターゲットと入力し、炎症を起こしたCNSに、その後、複数の細胞間に係合するように移植したNPCの能力に依存していることが示されている生体 11 内の特定の微小環境内で通信プログラム。特に中枢神経系を標的とするために、NPCは脳脊髄液（CSF）ICV注射による循環、または静脈内注射を介して血流への直接配信されます。一度血流またはCSFのいずれかを入力し、移植されたNPCは積極的に相互作用血液脳（BBB）または血液脳脊髄液（BCSFB）障壁とCNS実質を入力してください。 NPCグラフト及びBBB（又はBCSFB）との間のこの相互作用は、NPC表面の細胞接着分子（CAM）の特定のセットにより調節および活性化内皮/上衣細胞12-14上のCAMカウンターリガンドの高レベルの発現によって促進される。これらのCAMの例としては、ヒアルロン酸の受容体であるCD44、および細胞間接着分子（ICAM）-1のリガンドは非常に遅い抗原（VLA）-4 5,15,16（つまり、白血球では、活性化上衣との相互作用の責任があるが含まれおよび内皮細胞）、およびはるかに低い程度のリンパ球機能関連抗原（LFA）-1およびP-セレクチン糖タンパク質リガンド（PSGL）-1。 NPCはまた、CCR1、CCR2、CCR5、CXCR3、およびCXCR4を含む、ケモカイン受容体の広範囲を発現する（しかし、CCR3およびCCR7を発現しない）、 インビトロおよびインビボの両方で 5,16、機能的に活性である。このように、systemicaLLYのNPCは、炎症を起こしたCNSのレベルで蓄積する、Gタンパク質共役受容体（GPCRは）とともに、これらのCAMを使用して注入した。逆に、NPCは、血管や脳脊髄液の空間ルート2 を介して中枢神経系を入力しない健康なマウスに全身的に注射。 CNS炎症、または化学的に誘発された脳炎のモデルとして、全身性サイトカインまたはlypopolisaccharide（LPS）の注射後に内皮/上衣細胞の活性化は、脳や脊髄の2に全身に注入されたNPCの蓄積が必要である。このようにして、全身のNPC療法とCNSの成功ターゲティングは、脳と脊髄環境が蓄積とのNPCの経内皮移動に資するである、機会（ウー）の病気特定のウィンドウの識別に依存している。このような条件は、一般的に、急性および亜急性炎症17のコンテキストで発生する。一度、未分化のNPCを移植し、中枢神経系に入ったマウスの臨床病理学的な特徴だけでなく、EAEを持つ大規模、ヒト以外の霊長類を改善することが示されている。これはに応じて最小限の細胞置換2および非CNS炎症を起こした領域19,20（ 例えばリンパ節）VS血管周囲のCNS ​​2,5,6,18内の免疫調節および神経保護パラクリン因子の分泌が顕著で依存していることが記載されている炎症細胞シグナル伝達は、免疫細胞の浸潤を5により誘発される。 ここに我々は、慢性EAEのマウスモデルに、体のNPCの全身注射の主要な方法論の側面を説明します。具体的には、我々は我々が（i）に設置していたプロトコルを展開し、派生して、大人のC57BL / 6マウスの脳室下帯（SVZ）からの移植体細胞のNPCの準備を定義します。 （ii）は、マウスの慢性EAEを誘導し、（iii）の（ 静脈内または脳室内投与 ）治療的に有効な全身を行うNPC移植iをNTO EAEマウス。

<table border="0" cellpadding="0" cellspacing="0" width="1474">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:753px;">Cell culture</td>
			<td style="width:300px;"></td>
			<td style="width:149px;"></td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EBSS</td>
			<td>Sigma</td>
			<td>E2888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Cystein</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papain</td>
			<td>WORTHINGTON</td>
			<td>30H11965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher scientific</td>
			<td>D/0700/50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse NeuroCult basal medium</td>
			<td>Stem Cell technologies</td>
			<td>05700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult proliferation supplements</td>
			<td>Stem Cell technologies</td>
			<td>05701</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic fibroblast growth factor</td>
			<td>Peprotech</td>
			<td>100-18B-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal growth factor</td>
			<td>Peprotech</td>
			<td>AF-100-15-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td>1514012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel (coating solution)</td>
			<td>BD biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult&#174; Differentiation Kit (Mouse)</td>
			<td>Stem cell technologies</td>
			<td>05704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accumax</td>
			<td>eBioscience</td>
			<td>00-4666-56</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS (DPBS) (10x) without Ca&#38; Mg</td>
			<td>PAA LABORATORIES LTD</td>
			<td>H15-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Myco trace</td>
			<td>PAA LABORATORIES LTD</td>
			<td>Q052-020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>SIGMA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">immunofluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>PAA LABORATORIES LTD</td>
			<td>B11-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Nestin</td>
			<td>Abcam</td>
			<td>ab11306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti GFAP</td>
			<td>DAKO</td>
			<td>203344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Histone H3 (phospho S10)&#160;</td>
			<td>Abcam</td>
			<td>ab14955</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti MAP-2</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti MBP</td>
			<td>AbD SEROTEC</td>
			<td>MCA409S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-O4 Antibody, clone 81 | MAB345</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mounting solution</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EAE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freund&#39;s Adjuvant Incomplete</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mycobacterium tuberculosis&#160;</td>
			<td>DIFCO</td>
			<td>H37Ra</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOG(35&#8211;55)&#160;</td>
			<td>Espikem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pertussis toxin</td>
			<td>List Biological Laboratories</td>
			<td>181</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris scissor straight</td>
			<td>Fine Sciences Tolls</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blunt/bended forceps</td>
			<td>Fine Sciences Tolls</td>
			<td>11080-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Brain slicer</td>
			<td>Zivic instruments</td>
			<td>BSMAS005-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blades</td>
			<td>Swann-Morton</td>
			<td>324</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P200, P1000 pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine (Vetalar)</td>
			<td style="width:300px;">Boehringer Ingelheim</td>
			<td>01LC0030&#160;&#160;</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>32371</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereotaxic frame</td>
			<td>KOPF</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7762-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde (PFA)</td>
			<td>SIGMA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASTAIN Elite ABC Kit</td>
			<td>vector laboratories</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
	</tbody>
</table>

systemic injection of neural stem/progenitor cells in mice with chronic eae

神経幹/前駆細胞（NPCの）は、再生神経の脳の修復や復元を目的とした移植のアプローチのための有望な幹細胞源である。このディレクティブは、脳の修復が神経疾患のいくつかの前臨床モデルでの焦点または全身NPC移植後に達成されていることを豊富な証拠から生じている。 これらの実験データは、緊急の評価を必要とする脳疾患のための修復、幹細胞治療の主なハードルの一つとして細胞送達経路を同定した。実質内の幹細胞移植は、例えば、脊髄損傷およびパーキンソン病のような単離され、アクセス可能な脳の病変によって特徴付けられる病状の論理的なアプローチを表す。残念ながら、この原理は、多発性硬化症（MS）を含む、不十分、多焦点を特徴とする状態に適用炎症性および散在性（時間的にも空間の）性質である。このように、脳はシステム経験による標的EMIC NPC送達は、脳および中枢神経系（CNS）の実験的慢性炎症性損傷の影響を受け齧歯類およびヒト以外の霊長類の脊髄に細胞を送達するための低侵襲性治療上有効なプロトコルとなっている。 細胞送達のこの代替方法は、（i）に特異的にそれらの生得的な能力、NPCのpathotropismに依存している機能的な細胞接着分子および炎症性サイトカインおよびケモカイン受容体を介して環境を感知し; （II）静脈後漏れるの解剖学的障壁交差または脳室内（ICV）注射（ 静脈 ）; （iii）の炎症性の脳および脊髄損傷の複数の血管周囲部位（単数または複数）のレベルで蓄積する。および（iv）インビボで異なる宿主標的細胞上に顕著な組織栄養および免疫調節効果を発揮する。 ここでは、我々はIVのために開発した方法が記載されている。と&lt;EM慢性CNS炎症性脱髄のモデルとして実験的自己免疫性脳脊髄炎（EAE）マウスにおける同系のNPC&gt; ICV配信、および再生神経学における炎症を起こした脳の選択的標的化のための貴重な技術のような全身性幹細胞送達を想定。

NPCの派生と特性評価 SVZ解剖は、機械的および酵素的解離（ 図1A）によって6〜8週齢のC57Bl / 6マウスのた（n = 5-7マウス/プール）のプール上で行われる。 CGMにおける培養の数日後、自由に浮遊ニューロスフェアは、（ 図1Aおよび1B）を形成し始める。主な球を回収し、機械的に、すべての4-5 DIVを継代する。継代時には、生細胞および死細?...

Watch this Scientific Journal Video about Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE at JoVE.com

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

神経幹/前駆細胞（NPCの）の移植は、再生神経学に大きな約束を保持している。のNPCの全身送達は、脳および中枢神経系の実験的慢性炎症性損傷の影響を受け齧歯類およびヒト以外の霊長類の脊髄に幹細胞を提供するために効果的な、侵襲的な低、かつ治療に非常に効果的なプロトコルになっています。

慢性EAEを有するマウスにおける神経幹/前駆細胞の全身注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

systemic-injection-neural-stemprogenitor-cells-mice-with-chronic

Research

JoVE Journal

Immunology and Infection

17.5K ビュー.  University of Cambridge, UK. 神経幹/前駆細胞（NPCの）の移植は、再生神経学に大きな約束を保持している。のNPCの全身送達は、脳および中枢神経系の実験的慢性炎症性損傷の影響を受け齧歯類およびヒト以外の霊長類の脊髄に幹細胞を提供するために効果的な、侵襲的な低、かつ治療に非常に効果的なプロトコルになっています。 

ビデオ: Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

精製造血幹細胞/前駆細胞のDNAの変異を特定する

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

免疫・感染学

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

ミエリンオリゴデンドロサイト糖タンパク質（MOG<sub&gt; 35〜55</sub&gt;）C57BL / 6マウスにおける実験的自己免疫性脳脊髄炎（EAE）誘発

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

局所および全身所における病原体により誘導される慢性炎症のためのマウスモデル

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表現型および慢性リンパ球性脈絡髄膜炎ウイルスに感染したマウスから単離された活性化制御性T細胞の機能解析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用してリンパ球血管外漏出の分析<em&gt;インビトロ</em&gt;ヒト血液脳関門のモデル

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

眼球組織におけるエフェクター記憶T細胞の増加を伴う慢性自己免疫ドライアイラットモデル

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

グルコース-6-リン酸イソメラーゼ(G6PI)誘発RAマウスにおけるiNKT細胞の導入免疫療法

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

マウスの不感性リンパ管を縫合することによりリンパ流を遮断する

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. This requires methods for the direct deposition of reagents and/or cells into the thymus of living mice. Traditional methods of intrathymic injection include thoracic surgery or minimally invasive percutaneous blind injections, both of which have significant limitations. Ultra-high frequency ultrasound imaging devices have made image-guided percutaneous injections possible in mice, greatly improving the injection accuracy of the percutaneous injection approach and enabling the injection of smaller targets. However, image-guided injections rely on the utilization of an integrated rail system, making this a rigid and time-consuming procedure. A unique, safe, and efficient method for percutaneous intrathymic injections in mice is presented here, eliminating reliance on the rail system for injections. The technique relies on using a high-resolution micro-ultrasound unit to image the mouse thymus noninvasively. Using a free-hand technique, a radiologist can place a needle tip directly into the mouse thymus under sonographic guidance. Mice are cleaned and anesthetized before imaging. For an experienced radiologist adept at ultrasound-guided procedures, the learning period for the stated technique is quite short, typically within one session. The method has a low morbidity and mortality rate for the mice and is much faster than current mechanically assisted techniques for percutaneous injection. It allows the investigator to efficiently perform precise and reliable percutaneous injections of thymuses of any size (including very small organs such as the thymus of aged or immunodeficient mice) with minimal stress on the animal. This method enables the injection of individual lobes if desired and facilitates large-scale experiments due to the time-saving nature of the procedure.

The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections.

マウスの胸腺内注射のための低侵襲、正確、効率的な技術

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. ...