المؤلفين أشكر جايدن سميث لاستعراض نقدي وإثبات تحرير المخطوطة. وقد تلقى هذا العمل دعما من الجمعية الوطنية التصلب المتعدد (NMSS والمنح الجزئية RG-4001-A1)، والإيطالية جمعية التصلب المتعدد (AISM، منح 2010/R/31)، وزارة الصحة الإيطالية (GR08-7)، أجنحة من أجل الحياة، بنكا بوبولاري اجريكولا دي راغوزا (BAPR)، ومجلس البحوث الأوروبي (ERC) بموجب اتفاق ERC-2010-جنيها استرلينيا أي برنامج المنح الإطار 7 الجماعة الأوروبية (EC) 260511-SEM_SEM و(FP7/2007-2013) تحت اتفاقية المنحة ن * درجة؛ 280772 - حصرى.

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والكتاب ليس لديهم ما يكشف.

العلاج بالخلايا الجذعية الجسدية ومقرها الناشئة باعتبارها واحدة من أهم الاستراتيجيات الواعدة لعلاج الاضطرابات الالتهابية المزمنة CNS مثل MS2 11. في حين أن آليات الحفاظ على آثارها العلاجية لا تزال بحاجة إلى توضيح تماما، ونظرا لتأثير كبير من زرع مجلس الشعب في نماذج ...

نشأت أدلة قوية مستمدة من الدراسات المجراة مما يدل على الكفاءة العلاجية من زرع الخلايا الجذعية العصبية الجسدية / السلائف (الشخصيات) في النماذج الحيوانية من اضطرابات الجهاز العصبي المركزي 1-8. ومع ذلك، هناك عدد من القضايا المتعلقة إيصال الخلايا الجذعية إلى المضيف تتطلب دراسة متأنية قبل أن تترجم هذه النتائج التجريبية في التطبيقات السريرية. وهناك عقبة كبيرة لا سيما من أجل وضع (nonhematopoietic) التصالحية العلاج بالخلايا الجذعية لمتعددة البؤر، أمراض الدماغ المزمنة التهابات هو تحديد الطريق الأمثل لحقن مجلس الشعب. فهم شركة الفيزيولوجيا المرضية للمرض المستهدفة (تنسيق أو متعددة البؤر؛ الأولية التنكسية التهابات أو الأساسي)، وتحليل حذرا من القضايا الجدوى والمخاطر المرتبطة التقنيات تسليم في تحديد البروتوكول الأمثل للتسليم الخلايا الجذعية. في حين أن التنسيق ( <em&gt; على سبيل المثال. في لحمة الجهاز العصبي) زرع الخلايا الجذعية هو نهج منطقي لعلاج أمراض الجهاز العصبي المركزي تتميز المناطق المحصورة مكانيا من الضرر (مثل الشلل الرعاش ومرض هنتنغتون، والمخ والحبل الشوكي الإصابات، والسكتة الدماغية)، نفس النهج جدا قد يثبت أن يكون عمليا غير مجدية في ظروف مثل MS، حيث متعددة البؤر، المزمنة، ونشرها مكانيا ضرر CNS يتراكم مع مرور الوقت. في هذه الحالة الأخيرة، التي تستهدف حقن الخلايا تنسيق لآفات الفردية وأعاقت أيضا القدرة محدود من الشخصيات المزروعة على الهجرة لمسافات طويلة داخل لحمة الجهاز العصبي المركزي، مما أثار تحديد طرق بديلة، وأكثر مناسبة من الجهاز العصبي المركزي التي تستهدف زرع مع مجلس الشعب أقل الغازية . ظهرت عدا كبيرا من الملاحظات التي الشخصيات استهداف الورم داخل القحف (على سبيل المثال. دبقي) في الفئران عندما تم حقنها intravascularly خارج CNS9. تتبع هذا المنويةفي الجسم الحي من الأدلة pathotrophism الخلايا الجذعية 10، وقد تراكمت البيانات واسعة النطاق تتعلق الجدوى والنجاعة العلاجية للزرع النظامية من الشخصيات في الحيوانات المختبرية مع التهاب الدماغ التجريبية المناعة الذاتية (EAE)، كنموذج للالتهابات الضرر العصبي المركزي، إما عن طريق الوريد (د) أو intracerebroventricular (ICV). مجلس الشعب حقن 1،2،5،6،8 .. لقد عرض لأول مرة أن هذا يعتمد على القدرة على زرع لاستهداف الشخصيات وأدخل CNS ملتهبة، والدخول في وقت لاحق بين الخلايا متعددة برامج الاتصالات داخل microenvironments محددة في الجسم الحي 11. من أجل خصيصا لاستهداف الجهاز العصبي المركزي، ويتم تسليمها الشخصيات مباشرة في السائل النخاعي (CSF) الدورة الدموية عن طريق الحقن ICV، أو في مجرى الدم عن طريق الحقن د. مرة واحدة إما دخول مجرى الدم أو السائل النخاعي، الشخصيات زرع تتفاعل بنشاط معوالدم في الدماغ (BBB) ​​أو الدم السائل النخاعي (BCSFB) الحواجز وأدخل حمة الجهاز العصبي المركزي. وينظم هذا التفاعل بين مجلس الشعب والكسب غير المشروع BBB (أو BCSFB) من خلال مجموعة محددة من سطح مجلس الشعب جزيئات التصاق الخلية (الحدب) ويسر عن طريق التعبير عن مستويات عالية من CAM مكافحة بروابط على تنشيط البطانية / خلايا البطانة العصبية 12-14. أمثلة من هذه الحدب تشمل مستقبلات للهيالورونات، CD44، وجزيء التصاق بين الخلايا (ICAM) -1 يجند في وقت متأخر جدا المستضد (VLA) -4 5،15،16 (أنه في الكريات البيض، هي المسؤولة عن التفاعل مع البطانة العصبية تفعيلها والخلايا البطانية)، وإلى حد أقل بكثير اللمفاويات وظيفة المرتبطة مستضد (LFA) -1 و ف selectin بروتين سكري يجند (PSGL) -1. الشخصيات أيضا أن أعرب عن مجموعة واسعة من المستقبلات chemokine، بما في ذلك CCR1، CCR2، CCR5، CXCR3، وCXCR4 (ولكن لا تعبر عن CCR3 وCCR7)، والتي هي وظيفيا نشطة، سواء في المختبر والمجراة 5،16. وبالتالي، systemicaالشخصيات حقن LLY استخدام هذه الحدب، جنبا إلى جنب مع G-مستقبلات البروتين يقترن (GPCRs)، لتتراكم على مستوى الجهاز العصبي المركزي ملتهبة. على العكس، حقن الشخصيات بشكل منتظم في الفئران صحية لا تدخل الجهاز العصبي المركزي عن طريق الأوعية الدموية أو المسارات الفضاء السائل النخاعي 2. CNS التهاب، أو البطانية / تنشيط الخلايا البطانة العصبية التالية خلوى النظامية أو lypopolisaccharide (LPS) حقن كنموذج للالتهاب الدماغ الناجم عن كيميائيا، لذا فمن الضروري لتراكم الشخصيات حقن منتظم في الدماغ والحبل الشوكي 2. وبالتالي، والاستهداف الناجح لCNS مع العلاجات مجلس الشعب النظامية تعتمد على تحديد نافذة معينة المرض الفرص (وو) التي الدماغ والحبل الشوكي بيئة تفضي إلى تراكم والهجرة transendothelial من الشخصيات. تنشأ مثل هذه الظروف عادة في سياق الحاد وتحت الحاد التهاب 17. مرة واحدة بعد أن دخلت الجهاز العصبي المركزي، زرع الشخصيات غير متمايزةوقد تبين لتحسين ميزات الممارسة الطبية السريرية المرضية من الفئران وكذلك أكبر، الرئيسيات غير البشرية مع EAE. وقد وصفت هذه أن تعتمد الحد الأدنى من استبدال الخلايا وإفراز 2 ملحوظا من عوامل المناعة نظير الصماوي التنظيمية واعصاب داخل الجهاز العصبي المركزي حول الوعاء 2،5،6،18 مقابل غير CNS المناطق الملتهبة 19،20 (مثل العقد الليمفاوية) في استجابة لل إشارة الخلية التهابات التي تسببها تسلل الخلايا المناعية 5. هنا نحن تصف الجوانب المنهجية الأساسية لحقن النظامية من الشخصيات الجسدية في نموذج الفأر من EAE المزمن. وبشكل أكثر تحديدا، يمكننا تعريف البروتوكولات التي وضعناها ل(ط) اشتقاق، وتوسيع والاستعداد لزرع الشخصيات الجسدية من منطقة subventricular (SVZ) من الكبار C57BL / 6 الفئران؛ (ب) لحث على EAE المزمنة في مثل الفئران و (iii) أداء فعالة علاجيا النظامية (في الوريد أو ICV) ط زرع مجلس الشعبn لالفئران EAE.

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			<td height="21" style="height:21px;width:753px;">Cell culture</td>
			<td style="width:300px;"></td>
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			<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

الخلايا الجذعية / العصبية السلائف (الشخصيات) هي مصدر الخلايا الجذعية واعدة للمناهج تهدف إلى زرع إصلاح الدماغ أو ترميم الأعصاب في التجدد. وقد نشأ هذا التوجيه من الأدلة الوفيرة على أن يتحقق إصلاح الدماغ بعد تنسيق أو النظامية زرع مجلس الشعب في العديد من النماذج قبل السريرية للأمراض العصبية. وقد حددت هذه البيانات التجريبية الطريق تسليم خلية واحدة من العقبات الرئيسية لالتصالحية العلاج بالخلايا الجذعية لأمراض الدماغ التي تتطلب تقييم عاجلة. يمثل Intraparenchymal ترقيع الخلايا الجذعية نهج منطقي لتلك الأمراض التي تتميز آفات الدماغ معزولة ويمكن الوصول إليها مثل إصابات الحبل الشوكي ومرض باركنسون. للأسف، وهذا المبدأ ينطبق بشكل سيئ لظروف تتميز الطبيعة متعددة البؤر، التهابات ونشرها (سواء في الزمان والمكان)، بما في ذلك مرض التصلب المتعدد (MS). على هذا النحو، الدماغ استهداف SYSTأصبح EMIC تسليم مجلس الشعب منخفض بروتوكول الغازية وفعالة علاجيا لتقديم الخلايا إلى الدماغ والحبل الشوكي من القوارض والرئيسيات غير البشرية تتأثر التجريبية الضرر التهابات مزمنة في الجهاز العصبي المركزي (CNS). هذه الطريقة بديل من تسليم خلية يعتمد على توجه للنسيج المريض مجلس الشعب، وتحديدا القدرة الفطرية ل(ط) بمعنى البيئة وظيفية عبر جزيئات التصاق الخلية وخلوى وchemokine مستقبلات التهابات؛ (ب) عبور الحواجز التشريحية تسرب بعد الوريد (رابعا) أو intracerebroventricular (ICV) الحقن؛ (ج) تتراكم على مستوى الموقع حول الوعاء متعددة (ق) من التهابات الدماغ والنخاع الشوكي الضرر؛ و (iv) ممارسة التغذية الأنسجة ملحوظا والآثار التنظيمية المناعة على الخلايا المستهدفة المضيف مختلفة في الجسم الحي. نحن هنا وصف الطرق التي قمنا بتطويرها لد. و&lt;م&gt; تسليم ICV من الشخصيات مسانج في الفئران مع التهاب الدماغ التجريبية المناعة الذاتية (EAE)، ونموذج من الجهاز العصبي المركزي إزالة الميالين المزمن التهابات، وتتوخى تسليم خلية جذعية النظامية كأسلوب قيمة لاستهداف انتقائي من الدماغ ملتهبة في علم الأعصاب التجدد.

مجلس الشعب الاشتقاق وتوصيف يتم إجراء تشريح SVZ على برك (ن = 5-7 الفئران / حمام السباحة) من 6-8 الاسبوع القديمة C57BL / 6 الفئران عن طريق التفكك الميكانيكية والأنزيمية (الشكل 1A). بعد أيام قليلة من زراعة في CGM، neurospheres التعويم الحر يبدأ تشكيل (أرقام 1A و 1B).</...

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

زرع الخلايا الجذعية / السلف العصبية (الشخصيات) يحمل وعودا كبيرة في الأعصاب التجدد. تحولت تسليم النظامية من الشخصيات في انخفاض الغازية، وعلاجية فعالة جدا بروتوكول فعالة، لتقديم الخلايا الجذعية في الدماغ والحبل الشوكي من القوارض والرئيسيات غير البشرية تتأثر التجريبية الضرر التهابات مزمنة في الجهاز العصبي المركزي.

حقن النظامية من الخلايا الجذعية العصبية / السلف في الفئران مع EAE المزمن

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 Views.  University of Cambridge, UK.  زرع الخلايا الجذعية / السلف العصبية (الشخصيات) يحمل وعودا كبيرة في الأعصاب التجدد. تحولت تسليم النظامية من الشخصيات في انخفاض الغازية، وعلاجية فعالة جدا بروتوكول فعالة، لتقديم الخلايا الجذعية في الدماغ والحبل الشوكي من القوارض والرئيسيات...

Video: 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.

تحديد الطفرات الحمض النووي في الخلايا الجذعية المكونة للدم تنقية / السلف

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

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.

على المدى الطويل المزمن<em&gt; الزائفة الزنجارية</em&gt; عدوى مجرى الهواء في الفئران

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.

المايلين دبقية قليلة التغصن جلايكوبروتين (MOG<sub&gt; 35-55</sub&gt;) المستحثة التجريبية المناعة الذاتية التهاب الدماغ (EAE) في C57BL / 6 الفئران

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.

نموذج الفأر لالتهاب الناجم عن مسببات الأمراض المزمنة في مواقع المحلية والجهازية

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.

المظهرية والتحليل الوظيفي من المنشط خلايا تنظيم تي المعزولة من الفئران المصابة بفيروس المزمنة الليمفاوي المشيمي

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.

تحليل الخلايا الليمفاوية التسرب باستخدام<em&gt; في المختبر</em&gt; نموذج من الدم في الدماغ الإنسان الحاجز

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.

A المناعة الذاتية الجفاف العين الفئران نموذج مع زيادة في المفعول الذاكرة خلايا T في الأنسجة مقلة العين

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.

العلاج المناعي بالتبني من خلايا iNKT في الجلوكوز-6-الفوسفات Isomerase (G6PI) الناجمة عن الفئران RA

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.

منع تدفق اللمفاوية عن طريق خياطة الأوعية اللمفاوية Afferent في الفئران

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.

تقنية طفيفة التوغل ودقيقة وفعالة للحقن داخل الزعتر في الفئران

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