This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

بيان الأخلاق: تمت الموافقة على هذا البروتوكول من قبل لجنة جامعة ديوك المؤسسية رعاية الحيوان واستخدام ويتبع كل مبادئ توجيهية لاستخدام الأخلاقية للحيوانات. 1. إعداد معدات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأوتوكلاف الأدوات الجراحية قبل الجراحة. </li><li style=";text-align:right;direction:rtl"> تطهير الجهاز المجسم مع الايثانول 70٪. </li><li style=";text-align:right;direction:rtl"> بدوره على حمام المياه والحفاظ على درجة حرارة المياه على 42 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> حل نوع IV-S المطثية كولاجيناز في محلول ملحي بتركيز 0.075 U في 0.4 ميكرولتر. </li></ol> 2. كولاجيناز حقن نموذج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وزن الماوس. </li><li style=";text-align:right;direction:rtl"> تخدير الماوس في غرفة الاستقراء مع 5٪ في isoflurane و30٪ O 2/70٪ N 2. وأشار التخدير كافية بعد حوالي 2 دقيقة عندما تباطأ التنفس الماوس إلى 1 في الثانية الواحدة. </li><li style=";text-align:right;direction:rtl"> التنبيب القصبة الهوائية مع 30 ملم 20 G القسطرة الوريدية. </li><li style=";text-align:right;direction:rtl"> الاتصال القسطرة لالتنفس الصناعي القوارض وتهوية الرئتين ميكانيكيا مع 1.6٪ في isoflurane و30٪ O 2/70٪ N 2 بمعدل 105 نفسا في الدقيقة مع حجم المد والجزر تسليمها من 0.75 مل .. </li><li style=";text-align:right;direction:rtl"> يحلق فروة الرأس مع ماكينة حلاقة الإلكترونية. مرة واحدة يتم تخدير الماوس ومدخل أنبوب، نقله إلى محطة عمل مختلفة للحلاقة، ثم عاد إلى مقاعد البدلاء الجراحية. </li><li style=";text-align:right;direction:rtl"> تأمين الرأس في إطار المجسم، ومستوى الرأس مع خياطة كلا الاكليلية والسهمي كنقاط مرجعية. </li><li style=";text-align:right;direction:rtl"> تطبيق مرهم للعين للعيون. </li><li style=";text-align:right;direction:rtl"> إدراج التحقيق في درجة الحرارة المستقيم. الحفاظ على درجة حرارة الجسم في 37.0 ± 0.2 ° C باستخدام بانسيابية تعميم مائي. </li><li style=";text-align:right;direction:rtl"> مسح منطقة العمليات الجراحية مع بتدين اتباعها مع الايثانول 70٪ وكرر 3 مرات. </li><li style=";text-align:right;direction:rtl"> جعل 1 سم خط الوسط فروة الرأس شق ومسح السمحاق أفقيا مع القطن ذات الرؤوس قضيب العقيمة لفضح bregma. </li><li style=";text-align:right;direction:rtl"> الحفر 1 مم حفرة قطرها 2.2 مم لدغ غادر اللاتاألطراف إلى bregma مع حفر المياه المبردة. </li><li style=";text-align:right;direction:rtl"> تدوير القارورة كولاجيناز 5 مرات، ثم غسل حقنة 0.5 ميكرولتر مع 25 G إبرة (تعلق على الإطار المجسم) مع 0.5 ميكرولتر حل كولاجيناز 5 مرات (اترك 0.5 ميكرولتر من محلول كولاجيناز في حقنة بعد غسل الماضي). </li><li style=";text-align:right;direction:rtl"> محاذاة طرف الإبرة مع ثقب لدغ ثم طرد 0.1 ميكرولتر من حقنة ويمسح شطبة إبرة مع الحلاقة لتجاهل. </li><li style=";text-align:right;direction:rtl"> باستخدام مياداة مجهرية، ودفع الإبرة 3 ملم العميق للقشرة وترك بلا حراك لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> ضخ 0.4 ميكرولتر أكثر من 90 ثانية. </li><li style=";text-align:right;direction:rtl"> تقليل isoflurane وإلى 1٪ وترك بلا حراك إبرة لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> سحب الإبرة ببطء. </li><li style=";text-align:right;direction:rtl"> تطبيق 1-2 قطرات من 0.25٪ بوبيفاكايين تحت الجلد وخياطة الجلد. </li><li style=";text-align:right;direction:rtl"> إيقاف المرذاذ isoflurane وإزالة الماوس من إطار المجسم. </li><li style=";text-align:right;direction:rtl"> السماح الماوس لاستعادة التهوية عفوية مع نزع الأنبوب الرغامي اللاحقة. </li><li style=";text-align:right;direction:rtl"> عودة الماوس إلى القفص نظيفة والسماح بحرية الوصول إلىالغذاء والماء. </li></ol> 3. ذاتي حقن الدم نموذج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اتبع الخطوات 2،1-2،11 للنموذج حقن كولاجيناز. </li><li style=";text-align:right;direction:rtl"> رسم 50 ميكرولتر من محلول ملحي معقم الى ميكرولتر حقنة 30 G 50. </li><li style=";text-align:right;direction:rtl"> ربط حقنة ميكروليتر مع 70 سم أنبوب PE10. </li><li style=";text-align:right;direction:rtl"> طرد كل ملحي عادي من حقنة ميكروليتر في أنبوب PE10 تماما لإزالة أنابيب الهواء. </li><li style=";text-align:right;direction:rtl"> سحب المكبس حقنة ميكروليتر من 1 ملم إلى جعل فقاعة هواء في افتتاح البعيدة للPE10 أنبوب ميكروليتر جهاز حقنة لتجنب خليط من المياه المالحة والدم أثناء الإجراءات في وقت لاحق. </li><li style=";text-align:right;direction:rtl"> مسح القاصي الذيل وسط المنطقة الشريان من الفأر مع الايثانول 70٪، وقطع الشريان بشفرة الحلاقة عند 0.5 إلى 1 سم إلى طرف الذيل. </li><li style=";text-align:right;direction:rtl"> جمع 40 ميكرولتر من الدم من ذيل يقتطع من PE10 أنبوب ميكروليتر جهاز حقنة. ملاحظة: أن لا يتم استخدام الهيبارين في الإبرة، وأنابيب، أو الماوس. </li><li style=";text-align:right;direction:rtl"> نعلق حقنة ميكروليتر على injeمضخة كشن. </li><li style=";text-align:right;direction:rtl"> ربط المعدن قنية جزء من إبرة 27 G إلى نهاية الأنبوب PE10، وتأمين الإبرة إلى مياداة مجهرية على الإطار المجسم. </li><li style=";text-align:right;direction:rtl"> طرد 2 ميكرولتر من الدم من أصل 27 G الإبرة ومسح شطبة إبرة مع الحلاقة لتجاهل. </li><li style=";text-align:right;direction:rtl"> محاذاة طرف الإبرة مع ثقب إبرة لدغ وإدراج 3 ملم العميق للقشرة. </li><li style=";text-align:right;direction:rtl"> ضخ 35 ميكرولتر من الدم الذاتي بمعدل 2 ميكرولتر لكل دقيقة. </li><li style=";text-align:right;direction:rtl"> تقليل isoflurane وإلى 1٪ وترك بلا حراك إبرة لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> سحب الإبرة أكثر من 30 ثانية. </li><li style=";text-align:right;direction:rtl"> تطبيق 1-2 قطرات من 0.25٪ بوبيفاكايين تحت الجلد وخياطة الجلد. </li><li style=";text-align:right;direction:rtl"> إيقاف المرذاذ isoflurane وإزالة الماوس من إطار المجسم. </li><li style=";text-align:right;direction:rtl"> السماح الماوس لاستعادة التهوية عفوية مع نزع الأنبوب اللاحقة. </li><li style=";text-align:right;direction:rtl"> عودة الماوس إلى القفص نظيفة والسماح بحرية الوصول إلى الغذاء والماء. </li></ol> 4. عملية الشام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تتبع نفس الإجراءات لمباشرة صب كولاجينازنموذج ن، باستثناء من دون حقن إبرة بعد الإدراج. </li></ol> 5. رعاية ما بعد الجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حقن 0.5 مل تحت الجلد من الطبيعي المالحة في المساء لإجراء العمليات الجراحية في الجزء الخلفي من الرقبة الحيوان. </li><li style=";text-align:right;direction:rtl"> توفير الغذاء خففت بالماء وهلام الغذاء في أكواب بلاستيكية صغيرة توضع على أرضية القفص. استبدال المواد الغذائية يوميا لمدة 7 أيام. </li><li style=";text-align:right;direction:rtl"> تحقق من فقدان الوزن، والتئام الجروح، وعلامات الانزعاج يوميا لمدة 7 أيام. </li><li style=";text-align:right;direction:rtl"> إذا كانت هناك حاجة فترات انتعاش أكبر من 7 أيام، وإزالة خياطة يمكن أداؤها تحت ضوء التخدير استنشاق (حوالي 1٪ isoflurane وفي 30٪ O 2/70٪ N 2)، إذا لزم الأمر. </li></ol>

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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=A+mouse+model+of+intracerebral+hemorrhage+using+autologous+blood+infusion.">A mouse model of intracerebral hemorrhage using autologous blood infusion.</a> Nature Protocols. 3, 122-128 (2008).</li><li>Wang, J., Fields, J., Dore, 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+development+of+an+improved+preclinical+mouse+model+of+intracerebral+hemorrhage+using+double+infusion+of+autologous+whole+blood.">The development of an improved preclinical mouse model of intracerebral hemorrhage using double infusion of autologous whole blood.</a> Brain Research. 1222, 214-221 (2008).</li><li>MacLellan, C. L., 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=Intracerebral+hemorrhage+models+in+rat:+comparing+collagenase+to+blood+infusion.">Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion.</a> J Cereb Blood Flow Metab. 28, 516-525 (2008).</li></ol>

Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

على الرغم من البحوث قبل السريرية والتجارب السريرية الكبيرة الناتجة الناشئة للعلاجات واعدة 15-18، لا توجد التدخلات الدوائية أظهرت لتحسين النتيجة في ICH، ويبقى الرعاية الداعمة إلى حد كبير. قد يتم إنشاء قوائم العلاجات الممكنة بواسطة تكنولوجيات إنتاجية عالية، مثل ا?...

نزيف داخل المخ (ICH) هو شكل شائع نسبيا من الأمراض الدماغية الوعائية مع ما يقرب من 40-50٪ من المرضى المصابين يموتون في غضون 30 يوما 1. للأسف، أحرز تحسن طفيف في معدل وفيات خلال ال 20 سنة الماضية 2. وأكد تقارير من المعاهد الوطنية للصحة (3) والمبادئ التوجيهية من جمعية القلب الأمريكية 4 على أهمية تطوير النماذج ذات الصلة سريريا من ICH تمديد فهم الفيزيولوجيا المرضية ووضع أهداف للنهج علاجية جديدة. وتوجد عدة نماذج لمحاكاة ICH الإنسان 5. كما فهم ICH نضوج الفيزيولوجيا المرضية، فقد أصبح من الواضح أن مجموعة متنوعة من النماذج التي يمكن استخدامها لدراسة جوانب مختلفة من هذا المرض. وتشمل النماذج المستخدمة سابقا الفئران اميلويد اعتلال وعائي 6، intraparenchymal الإدراج microballoon والتضخم 7، والدم الشرياني المباشرتسلل 8،9. تم غرار فصي نزف من اميلويد اعتلال وعائي مع استخدام الفئران المعدلة وراثيا، ويمثل التراث الثقافي غير المادي سلالة متميزة. نماذج تحاكي تأثير Microballoon الشامل الحاد من تشكيل ورم دموي لكنه يفشل في التقاط استجابة الدماغ الخلوية إلى وجود الدم. أخيرا، مباشرة تسلل الدم الشرياني يخضع الدماغ للضغوط الشرياني من الشريان الفخذي. وهكذا، وهذا يحاكي نموذج الضغوط الشرياني ووجود الدم ولكن لا تعرض الدماغ لإصابة تمزق الاوعية الدموية الدقيقة من الأوعية الدموية الصغيرة. علاوة على ذلك، هذا النموذج قد تقلب عالية بطبيعتها. ومن المثير للاهتمام، الفئران ارتفاع ضغط الدم بشكل عفوي 10 تطوير عفوية ICH مع تقدمهم في السن. دراسة هذه الحيوانات بعد تطوير ICH قد تحاكي المرض في وجود واحد من الأمراض المصاحبة الرئيسية المهيئة للتراث الثقافي غير المادي البشر. في حين توجد هذه النماذج الأخرى، حقن intrastriatal من اللاهوائية كولاجيناز 11 أو حقن instrastiatal لالدم الكامل utologous 12 هي، في الوقت الراهن، واثنين من النماذج الأكثر شيوعا التي تستخدم في البحوث قبل السريرية ICH. وينبغي بذل ICH اختيار نموذج استنادا إلى الهدف من السؤال التجريبية، بما في ذلك اختيار الأنواع وطريقة حمل تشكيل دموي. على سبيل المثال، الخنازير هي الحيوانات الكبيرة مع وحدات التخزين بيضاء الدماغ المسألة كبيرة نسبيا بالمقارنة مع الفئران. وبالتالي، هي مناسبة نماذج الخنازير لدراسة المسألة الأبيض الفيزيولوجيا المرضية التالية ICH. في المقابل، أدمغة القوارض هي المادة الرمادية إلى حد كبير، ولكن الأنظمة المعدلة وراثيا جعل القوارض مفيدة لتقييم الآليات الجزيئية من الإصابة والتعافي بعد ICH. يحتوي كل نموذج على نقاط القوة والضعف المتأصلة فيها (الجدول 1)، والتي ينبغي النظر فيها بعناية قبل التجريب. البروتوكولات التالية تثبت الدم والحقن كولاجيناز نماذج ذاتي في الفئران. وقد ترجمت كل هذه النماذج من نماذج وضعت أصلا في الفئران13،14 والسماح باستخدام التكنولوجيا المعدلة وراثيا على نطاق واسع لاستكشاف الآليات الجزيئية المرتبطة موت الخلايا بعد ICH. سواء تمثل آليات الإصابة تختلف اختلافا واضحا من ICH الإنسان، وكلاهما النتيجة المتوقعة تختلف اختلافا واضحا من حيث التدابير السلوكية والنسيجية. وبالتالي، قد فرضيات معينة تصلح لنموذج واحد على الآخر، ولكن قد تتطلب التحقق من صحة العديد من الأفكار في كلا النموذجين. الجدول 1. مقارنة بين خصائص كولاجيناز وحقن الدم نزيف داخل المخ نماذج ذاتي. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td style="width:197px;"></td><td style="width:80px;"> كولاجيناز حقن </td><td style="width:66px;"> حقن الدم </td></tr><tr><td style="width:197px;"> سهولة الاستخدام </td><td style="width:80px;"> + + + </td><td style="width:66px;"> + + </td></tr><tr&gt; <td style="width:197px;"> استنساخ </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> السيطرة على النزيف الحجم </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + + </td></tr><tr><td style="width:197px;"> الجزر الدم </td><td style="width:80px;"> + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> يحاكي الأمراض البشرية </td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;"> بساطة </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> استخدام بأنواع متعددة </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

نزيف داخل المخ (ICH) هو شكل شائع من الأمراض الدماغية الوعائية ويترافق مع معدلات الاعتلال والوفيات كبيرة. نقص العلاج الفعال وفشل التجارب السريرية الكبيرة التي تستهدف الارقاء وإزالة جلطة تظهر الحاجة لمزيد من التحقيق يحركها آلية ICH. قد يتم تنفيذ هذا البحث من خلال الإطار الذي توفره نماذج قبل السريرية. وتشمل نموذجين الفئران في استخدام شعبية intrastriatal (العقد القاعدية) حقن الدم كله إما ذاتي أو كولاجيناز المطثية. منذ ذلك الحين، كل نموذج يمثل الميزات الفيزيولوجية المرضية تختلف اختلافا واضحا المتعلقة ICH، يمكن اختيار استخدام نموذج معين بناء على ما الجانب من المرض لدراستها. على سبيل المثال، وحقن الدم الذاتي يمثل أكثر دقة استجابة الدماغ على وجود الدم intraparenchymal، ويمكن تكرار أوثق فصي النزف. المطثية حقن كولاجيناز يمثل أكثر دقة لياليتمزق الأوعية مول وتطور ورم دموي سمات نزيف عميق. وبالتالي، فإن كل نتائج النموذج في تشكيل مختلف ورم دموي، والاستجابة neuroinflammatory والتنمية وذمة دماغية، ونتائج السلوك العصبي. قوة من التدخل العلاجي المزعوم يمكن أن يكون أفضل تقييمها باستخدام كلا النموذجين. في هذا البروتوكول، وأظهرت تحريض ICH باستخدام كلا النموذجين، مظاهرة بعد الجراحة الفورية للإصابة، وتقنيات الرعاية اللاحقة للعمليات الجراحية في وقت مبكر. كلا النموذجين تؤدي الى سقوط ضحايا استنساخه، وحجم ورم دموي، والعجز السلوك العصبي. بسبب عدم تجانس ICH الإنسان، وهناك حاجة إلى نماذج متعددة قبل السريرية لاستكشاف آليات الفسيولوجية المرضية بدقة واختبار الاستراتيجيات العلاجية المحتملة.

بسبب الاختلافات في تشكيل الورم الدموي (الشكل 1)، يظهر تحول المماثل مباشرة بعد الاستيقاظ بالنسبة للفئران حقنت الدم الذاتي وضمن 2-4 ساعة بعد الحقن كولاجيناز، كما يحدث توسع الورم الدموي (الشكل 2). غياب تحول المماثل يجب أن يثير القلق لعدم وجود إصابات كبير?...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

حقن Intrastriatal من ذاتي الدم أو اللاهوائية كولاجيناز كنماذج الفئران من نزف الدماغي الداخلي

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

intrastriatal-injection-autologous-blood-or-clostridial-collagenase

Research

JoVE Journal

Medicine

14.6K Views.  Duke University. Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase. 

Video: Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

حجم ثابت أو ضغط ثابت : نموذج الفئران من الصدمة النزفية

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

الشريان التاجي ربط Intramyocardial وحقن الفئران في نموذج من احتشاء

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

نزف الدماغي الداخلي النمذجة في الفئران: حقن الدم الذاتي أو البكتيرية كولاجيناز

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

A نموذج من الفئران تحت العنكبوتية نزف

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Stereological والتدفق الخلوي توصيف خلايا الدم البيضاء القطعان في نماذج من عابر أو الشلل الدائم نقص التروية

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

الأرنب نموذج الدم تحويلة لدراسة الحاد وعقابيل في وقت متأخر نزيف تحت العنكبوتية: الجوانب التقنية

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

الحديديك كلوريد الناجم عن الفئران التخثر نماذج

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

حقن إينتراكاردياك الموجهة بالموجات فوق الصوتية البشرية الخلايا الجذعية الوسيطة لزيادة صاروخ موجه للاستخدام في نماذج مورين من أمراض التهاب الأمعاء التجريبية للامعاء

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

العصب استرشادا مشجعا حقن الخلايا الجذعية الذاتي قرب عصب حنجري المتكررة الأيسر الخيلي

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

ثقب الوريد Subclavian كطريقة بديلة لجمع عينات الدم في الفئران

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...