The authors would like to thank all members of the Colucci lab for helpful discussions. This work was funded by The Wellcome Trust [094073/Z/10/Z], The Centre for Trophoblast Research and The British Heart Foundation.

<ol>
	<li>Burton, G. J., Woods, A. W., Jauniaux, E., Kingdom, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+and+physiological+consequences+of+conversion+of+the+maternal+spiral+arteries+for+uteroplacental+blood+flow+during+human+pregnancy.">Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy.</a> Placenta. 30 (6), 473-482 (2009).</li><li>Pijnenborg, R., Vercruysse, L., Hanssens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uterine+spiral+arteries+in+human+pregnancy:+facts+and+controversies.">The uterine spiral arteries in human pregnancy: facts and controversies.</a> Placenta. 27 (9-10), 939-958 (2006).</li><li>Ashkar, A. A., Croy, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-gamma+contributes+to+the+normalcy+of+murine+pregnancy.">Interferon-gamma contributes to the normalcy of murine pregnancy.</a> Biol Reprod. 61 (2), 493-502 (1999).</li><li>Ashkar, A. A., Croy, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+uterine+natural+killer+cells+are+mediated+by+interferon+gamma+production+during+murine+pregnancy.">Functions of uterine natural killer cells are mediated by interferon gamma production during murine pregnancy.</a> Semin Immunol. 13 (4), 235-241 (2001).</li><li>Ashkar, A. A., Di Santo, J. P., Croy, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+gamma+contributes+to+initiation+of+uterine+vascular+modification,+decidual+integrity,+and+uterine+natural+killer+cell+maturation+during+normal+murine+pregnancy.">Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy.</a> J Exp Med. 192 (2), 259-270 (2000).</li><li>Ashkar, A. 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=Assessment+of+requirements+for+IL-15+and+IFN+regulatory+factors+in+uterine+NK+cell+differentiation+and+function+during+pregnancy.">Assessment of requirements for IL-15 and IFN regulatory factors in uterine NK cell differentiation and function during pregnancy.</a> J Immunol. 171 (6), 2937-2944 (2003).</li><li>Barber, E. M., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uterine+NK+cell+population+requires+IL-15+but+these+cells+are+not+required+for+pregnancy+nor+the+resolution+of+a+Listeria+monocytogenes+infection.">The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection.</a> J Immunol. 171 (1), 37-46 (2003).</li><li>Colucci, F., Boulenouar, S., Kieckbusch, J., Moffett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+variability+of+immune+system+genes+affect+placentation.">How does variability of immune system genes affect placentation.</a> Placenta. 32 (8), 539-545 (2011).</li><li>Pijnenborg, R., Vercruysse, L., Brosens, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+placentation.">Deep placentation.</a> Best Pract Res Clin Obstet Gynaecol. 25 (3), 273-285 (2011).</li><li>Colucci, F., Kieckbusch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+uterine+natural+killer+cells+nurture+fetal+growth:+in+medio+stat+virtus.">Maternal uterine natural killer cells nurture fetal growth: in medio stat virtus.</a> Trends Mol Med. 21 (2), 60-67 (2015).</li><li>Hanna, J., 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=Decidual+NK+cells+regulate+key+developmental+processes+at+the+human+fetal-maternal+interface.">Decidual NK cells regulate key developmental processes at the human fetal-maternal interface.</a> Nat Med. 12 (9), 1065-1074 (2006).</li><li>Xiong, 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=Maternal+uterine+NK+cell-activating+receptor+KIR2DS1+enhances+placentation.">Maternal uterine NK cell-activating receptor KIR2DS1 enhances placentation.</a> J Clin Invest. 123 (10), 4264-4272 (2013).</li><li>Croy, B. A., Zhang, J., Tayade, C., Colucci, F., Yadi, H., Yamada, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+uterine+natural+killer+cells+in+mice.">Analysis of uterine natural killer cells in mice.</a> Methods Mol Biol. 612, 465-503 (2010).</li><li>Kieckbusch, J., Gaynor, L. M., Moffett, A., Colucci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC-dependent+inhibition+of+uterine+NK+cells+impedes+fetal+growth+and+decidual+vascular+remodelling.">MHC-dependent inhibition of uterine NK cells impedes fetal growth and decidual vascular remodelling.</a> Nat Commun. 5, (2014).</li><li>Zhang, J., Chen, Z., Smith, G. N., Croy, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cell-triggered+vascular+transformation:+maternal+care+before+birth.">Natural killer cell-triggered vascular transformation: maternal care before birth.</a> Cell Mol Immunol. 8 (1), 1-11 (2010).</li><li>Adamson, S. 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=Interactions+between+trophoblast+cells+and+the+maternal+and+fetal+circulation+in+the+mouse+placenta.">Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta.</a> Dev Biol. 250 (2), 358-373 (2002).</li><li>Colucci, F., Soudais, C., Rosmaraki, E., Vanes, L., Tybulewicz, V. L. J., Di Santo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+NK+cell+development+using+a+novel+alymphoid+mouse+model:+Investigating+the+role+of+the+c-abl+proto-oncogene+in+murine+NK+cell+differentiation.">Dissecting NK cell development using a novel alymphoid mouse model: Investigating the role of the c-abl proto-oncogene in murine NK cell differentiation.</a> J Immunol. 162 (5), 2761-2765 (1999).</li><li>Hu, D., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+Tpbpa-positive+trophoblast+precursors+leads+to+defects+in+maternal+spiral+artery+remodeling+in+the+mouse+placenta.">Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta.</a> Dev Biol. 358 (1), 231-239 (2011).</li><li>Zhang, J. H., Croy, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Ultrasonography+to+Define+Fetal-Maternal+Relationships:+Moving+from+Humans+to+Mice.">Using Ultrasonography to Define Fetal-Maternal Relationships: Moving from Humans to Mice.</a> Comparative Med. 59 (6), 527-533 (2009).</li></ol>

The authors have nothing to disclose.

تم تصميم بروتوكول المعروضة هنا لتوفير طريقة استنساخه التي يمكن من خلالها تقييم التغيرات الأوعية الدموية والتي هي ضرورية لنجاح الحمل. حاسمة لنجاح هذا التقييم هو نوعية المقاطع الأنسجة. كل من تحديد دقيق لعمر الحمل من المواقع الزرع وكذلك ligating موثوق شرايين الرحم هي خطوات حاسمة. التغييرات في المعلمات مثل وقت التثبيت، وبروتوكول معالجة الأنسجة، وما إلى ذلك قد يؤثر أيضا على النتيجة. لتقييم stereological غير منحازة، فمن المهم لقياس نفس العدد من السفن في كل موقع الزرع. فمن المستحسن لقياس 5 سفن في موقع غرس وزرع كل موقع في ثلاث نسخ. وتعني هذه القياسات 15 يمثل حجم متوسط ​​من أكبر 5 سفن. وبالإضافة إلى البيانات من Rag2 alymphoid - / - IL2rg - / - الفئران المعروضة هنا، وجدنا هذا البروتوكول الاضافيocol مفيدا لعدد من نماذج عدم كفاية الوظيفة المناعية الأمهات، بما في ذلك واحد من الخلايا تحول دون NK 14. كما تبين وجود عيوب في إعادة الشرايين في نماذج من ضعاف غزو الأرومة الغاذية 18 و الأساليب المذكورة هنا قد تكون مفيدة لتقييم الأوعية الدموية في هذه الحالات. نحن الأمثل والتحقق من صحة هذا البروتوكول للتحقيق في ساقطي إعادة الأوعية الدموية في منتصف الحمل في الماوس، وإنما هو أيضا تصور للتكيف مع هذا البروتوكول للعمل في أنظمة أخرى نموذج (مثل الفئران) أو حتى في غيرها من الأجهزة. في حين نجد أن إعادة NK-تعتمد يمكن تقييمها بقوة في gd9.5، فإنه يجوز تعديل للحصول على نقاط زمنية أخرى مثل gd12.5، عندما يتم تشكيلها الأوعية الدموية الأمهات تماما. عند هذه النقطة الزمن، غزو الأرومة الغاذية اللف هو أعمق 16 و قد تكون هذه النقطة وقت أكثر مناسبة للتحقيق في دور الأرومة الغاذية للتغيرات في الأوعية الدموية. وإذا رغبت قراءات كمية إضافية،يمكن للمحققين أيضا أن يزيد من عدد الأقسام الملون لSMA وتقييم نسبة السفن تشكيلها جزئيا داخل كل موقع الزرع. وجود قيود على هذا البروتوكول هو طبيعة وصفية الامتحانات النسيجية. المقايسات الفنية، ومع ذلك، يتم فقط أصبحت ببطء المتاحة (مثل دوبلر بالموجات فوق الصوتية 19). هذه التقنيات تتطلب خبرة فنية ونفقات أعلى بكثير للحصول على المعدات وصيانتها، ولكن لديها ميزة توفير طولية في الجسم الحي قراءات لتأثير التغيرات التي يمكن ملاحظتها تشريحيا. والعيب ممكن لجميع الامتحانات stereological هو الذاتية للمحققين. تحقيقا لهذه الغاية، وذلك باستخدام اثنين من الممتحنين مستقل لتحليل جميع العينات بشكل مستقل يمكن أن تساعد على تجنب هذه المشكلة. في حين أن البروتوكول المذكورة هنا يعطي نظرة ثاقبة كيف الكثير من الدماء يمكن أن تصل إلى واجهة الدم الجنيني للأم، قد يكون من المفيد أن الجمع بين ذلك مع مجمعات للنهج ementary تقييم المبلغ الإجمالي من الدم في أي وقت من الأوقات في الأوعية الدموية من خلال البلاستيك يلقي 16. قوة من هذا البروتوكول هو أنه يجمع بين نهجين مستقلة. وأول تقييم التغيير الأوعية الدموية كميا بواسطة التجسيم ومن ثم يتم التحقق من صحة نتيجة نوعيا باستخدام المناعية. موثوقية النتائج التي يمكن زيادة تعزيز العشوائي للعينات. يمكن بسهولة العينات التي أعدت لهذا البروتوكول أن تستخدم أيضا للتحقيق في غيرها من المعالم الحمل مثل decidualisation والتنمية من مجموع اللمفاوية mesometrial من الحمل (MLAp) أو مناعي من خلايا الفائدة، لتقييم صارم النمط الظاهري الحمل في midgestation. إعادة الشرياني هو خطوة حاسمة لحالات الحمل غير معقدة في النساء وفشل هذه التغييرات هي أساس المتلازمات كبيرة التوليد (السودانية)، وهي ما قبل تسمم الحمل، وتقييد نمو الجنين وولادة جنين ميت. نقدم هنا تقنيات لتقييم بعناية النمط الظاهري الحمل في نماذج الماوس التي تحاكي بعض خصائص الفيزيولوجيا المرضية لحكومة السودان.

وتوسط في تبادل المواد الغذائية والغازات والنفايات أثناء الحمل eutherian عن طريق المشيمة. في الجهاز التناسلي للأنثى، وشرايين الرحم هي القنوات الرئيسية من الدم إلى الرحم. بعد الزرع، هذه الفروع إلى سفن متخصصة تسمى الشرايين الحلزونية التي فائف من خلال القاعدي الساقط نحو وحدة جنيني مشيمي. قطر ومرونة هذه الشرايين الحلزونية، التي تحيط بها الكريات البيض، وبخاصة القاتلة الطبيعية الرحم (UNK) الخلايا، تملي حجم وسرعة الدم المتاح للالمشيمة 1،2. تغييرات الدورة الدموية الصحيحة نتيجة لإعادة الشرايين الحلزونية هي حاسمة لنجاح الحمل. في حين أن الآليات الكامنة وراء تختلف في التفاصيل بين الإنسان والحمل الفئران، والنتيجة النهائية لعملية إعادة عرض في كلا النوعين هي المتوسعة، وارتفاع الأوعية الدموية تصرف أن يفقد طبقة من العضلات الملساء. في الفئران، UNK الإنترفيرون الخلايا المشتقة من (IFN-) γ ضروري للحث على هذه التغييرات في حوالي منتصف الحمل 3-5. الفئران التي تفتقر إما خلايا NK أو مكونات IFN-γ يشير مسار تفشل للخضوع لهذه التغييرات وهذا يترافق مع انخفاض 6،7 نمو الجنين، مشددا على أهمية وجود تدفق الدم الكافي إلى وحدة جنيني مشيمي. خلال فترة الحمل البشري في وقت مبكر، وغزو الخلالي واللف من الأرومة الغاذية extravillous وتفاعلها مع الخلايا UNK مهمة لإحداث تغييرات الأوعية الدموية وتعزيز 10/8 نمو الجنين. خلايا UNK يمكن أن تنسق غزو الأرومة الغاذية البشري من خلال كيموكينات 11 والسيتوكينات مثل محببة بلعم - مستعمرة عامل تحفيز (GM-CSF) 12. ويرتبط غزو الأرومة الغاذية الضحلة مع انخفاض إعادة الشرايين ومضاعفات الحمل مثل تسمم الحمل 9. ويستند هذا البروتوكول على العمل الرائد آن كروي الذي وصف مهمةدور جهاز المناعة، وبخاصة NK المستمدة من IFN-γ لنجاح إعادة الأوعية الدموية من خلال المناعية ونقل التجارب أنيقة 5،13. نحن هنا وصف طريقة سريعة واقتصادية لتقييم حالة إعادة الشرايين الحلزونية. على الرغم من مختبرنا يقيم هذا بشكل روتيني في منتصف الحمل (الحمل اليوم (GD) 9.5) 14، فإن عملية إعادة تجري بين gd8.5 و 12.5 15. ظهور الماسحات الضوئية الشريحة الآلي سهلت كثيرا عملية تقييم المناطق السفينة والتجويف من أقسام ووجدنا أن يكون نهجا أكثر استنساخه من قياس أقطار السفينة. إضافة كشف المناعى من SMA تسمح التحقق من صحة واضحة للنتائج التي تم الحصول عليها من تقييم stereological. كما هو الحال مع جميع التقنيات stereological، فمن المستحسن لأداء تقييم كتجربة أعمى من قبل اثنين على الأقل الممتحنين. تحقيقا لهذه الغاية، ويمكن إدخال خطوة العشوائي عندما اخترق متسلسلتينغ العينات بحيث المحققين تقييم الشرائح لا يعرفون من الذي المجموعة التجريبية العينات تأتي. الهدف العام من هذا البروتوكول هو توفير أداة يمكن الاعتماد عليها لتقييم دقيق لإعادة الشرايين الحلزونية في الفئران مع تعريف الأنماط الجينية الأم والأب، في سياق نماذج الماوس لمضاعفات متعلقة بالحمل. النتائج تؤكد الاعتماد على الخلايا UNK من هذه العملية، وهو أمر ضروري لنمو الجنين الطبيعي.

<table border="0" cellpadding="0" cellspacing="0" width="704">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">C57BL/6 mice (8-12 weeks old)</td>
			<td style="width:99px;">Charles River</td>
			<td style="width:111px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Dental floss</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Polystyrene</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td>Used for holding tissue in place</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Straight dissection scissors</td>
			<td style="width:99px;">EMS</td>
			<td style="width:111px;">72940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">25G needles</td>
			<td style="width:99px;">BD&#160;</td>
			<td style="width:111px;">300600</td>
			<td>Used for holding tissue in place</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">50 ml conical tubes</td>
			<td style="width:99px;">Falcon</td>
			<td style="width:111px;">352070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Phosphate buffered saline</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">P3813-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Ethanol</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">32221-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Paraffin</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">327204-1KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Xylene</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:111px;">X/0250/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Automated tissue processor</td>
			<td style="width:99px;">Sakura</td>
			<td style="width:111px;">6032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Microtome</td>
			<td style="width:99px;">Leica</td>
			<td style="width:111px;">RM2245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Slide rack</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">Z710989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Coplin jar</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">S5891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Purified water</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Hematoxylin solution, Gill No. 3</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">GHS332-1L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Eosin Y solution</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">HT110116-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Fume hood</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Xylene-based mounting media</td>
			<td style="width:99px;">VWR</td>
			<td style="width:111px;">361254D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Slide scanner</td>
			<td style="width:99px;">Hamamatsu</td>
			<td style="width:111px;">NanoZoomer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Slide scanner software</td>
			<td style="width:99px;">Hamamatsu</td>
			<td style="width:111px;">NDP viewer</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Sodium citrate tribasic dihydrate</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">32320</td>
			<td>Make 10 mM solution and adjust pH to 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Pressure cooker</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td>Used for antigen retrieval</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Heating plate</td>
			<td style="width:99px;"></td>
			<td style="width:111px;"></td>
			<td>Used for antigen retrieval</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Hydrophobic barrier pen</td>
			<td style="width:99px;">Vector</td>
			<td style="width:111px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Hydrogen peroxide</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:111px;">H/1800/15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Tris buffered saline</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">T6664-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Mouse on mouse kit</td>
			<td style="width:99px;">Vector</td>
			<td style="width:111px;">BMK-2202</td>
			<td>Significantly reduces background</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Mouse anti-human smooth muscle actin</td>
			<td style="width:99px;">DAKO</td>
			<td style="width:111px;">M0851</td>
			<td>Cross-reacts with mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">IgG2a isotype control</td>
			<td style="width:99px;">DAKO</td>
			<td style="width:111px;">X0943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Humidified chamber</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">H6644</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">3,3&#8217;-Diaminobenzidine solution</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:111px;">D4293-5SET</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Hydrochloric acid</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:111px;">H/1050/PB17</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of maternal vascular remodeling during pregnancy in the mouse uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

Rag2 - / - IL2rg - / - الفئران تفتقر إلى جميع الخلايا الليمفاوية ناضجة، بما في ذلك الخلايا NK 17، وفشل شرايين الرحم لتخضع لتغيرات الأوعية الدموية ينظر في wildtype مسانج C57BL / 6 (WT) الفئران في منتصف الحمل 5،14. ونتيجة لهذا خفض Rag2 إعادة عرض - / - IL2rg - / - أظهرت الفئران مناطق أصغر بكثير اللمعية السطح، مما يدل على السفن عموما الصغيرة التي يمكن تصور على أقسام H &amp; E الملون وكميا stereologically (الشكل 2). وعلاوة على ذلك، وسمك الجدار النسبي (سفينة إلى نسبة التجويف) هو أعلى في الشرايين الحلزونية في هذه الفئران، مما يشير إلى انخفاض حجم المعروض من الدم وارتفاع سرعة الدم. وقد أكد هذا التقييم الكمي باستخدام كشف المناعى من SMA. ومن خصائص للفئران WT لتفقد أكثر من SMA في وسائل الاعلام الأوعية الدمويةالشرايين الحلزونية في منتصف الحمل. إذا لم هذه العملية يحركها الخلايا القاتلة الطبيعية تأخذ مكان، لا يزال المتوسط ​​المتحرك في وسائل الإعلام من السفن، ويمكن أن يتم الكشف بسهولة immunohistochemically كما عصابات حول الأوعية الدموية (الشكل 3). <img alt="الشكل 1" src="/files/ftp_upload/53534/53534fig1.jpg" /> الشكل 1: الفأر الرحم في gd9.5، نظرة عامة التخطيطي الرسم تبين المواقع النسبية للقرون الرحم وعنق الرحم (الأزرق) وشرايين الرحم الرئيسية (الأحمر). وأشار هي محور طويل من قرون الرحم (الأسهم) ونقطة ربط (السهام متقطع). يتم قطع الفروع لالمناعية على طول الطائرة للعرض. <img alt="الرقم 2" src="/files/ftp_upload/53534/53534fig2.jpg" /> الشكل 2: تقييم Stereological من إعادة الشرايين في منتصف الحمل (A) مثال ASSEssment من اللمعية ومجموع المناطق السفينة على أقسام H &amp; E الملون من الإناث WT في وسط 2/4 من القاعدي الساقط (رسمتها الأسود الرأسي الخطوط المتقطعة) في gd9.5. في السفن في أعلى التكبير، تحطمت على الخط الأحمر ترسم المساحة الإجمالية للسفينة، أصفر خط متقطع ترسم منطقة التجويف. بار = 500 ميكرون (يسار)، 50 ميكرون (يمين). المحور الطويل للرحم هو خط أفقي في هذا التوجه. (B) الكمي من المناطق اللمعية (اللوحة اليسرى) ونسبة المساحة اللمعية إلى مجموع مساحة الأوعية الدموية (اللوحة اليمنى) من WT (C57BL / 6) وRag2 - / - Il2rg - / - الفئران (على C57BL / 6 الخلفية). كل نقطة البيانات تمثل متوسط ​​15 القياسات (5 القياسات في القسم، 3 أقسام لكل موقع الزرع). الحانات تمثل يعني ن = 11-13 المواقع زرع من 4 حالات الحمل في الجماعة؛ ف قيمة من الذيل يومين، أونبايريد اختبار مان ويتني. WT: wildtype. <img alر = &quot;الشكل 3&quot; SRC = &quot;/ ملفات / ftp_upload / 53534 / 53534fig3.jpg&quot; /&gt; الرقم 3: كشف المناعى من الأكتين العضلات الملساء في القاعدي الساقط الممثل SMA تلطيخ على WT (أعلى) وRag2 - / - IL2rg - / - الفئران (على C57BL / 6 الخلفية، أسفل) في gd9.5. بار = 100 ميكرون. WT: wildtype.

Watch this Scientific Journal Video about Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus at JoVE.com

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

تقييم الأم الأوعية الدموية إعادة عرض أثناء الحمل في الرحم ماوس

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

assessment-maternal-vascular-remodeling-during-pregnancy-mouse

Research

JoVE Journal

Developmental Biology

13.1K Views.  University of Cambridge. This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry. 

Video: Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

التلاعب وظيفي من الأم جين المنتجات التي تستخدم<em&gt; في المختبر</em&gt; نضوج البويضة في الزرد

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

تحليل الناجم عن حمض الريتينويك العصبية تمايز الخلايا الجذعية الجنينية الماوس في الهيئات مضغي اثنين وثلاثة الأبعاد

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

تقنية زرع الثدي الثديية الرواية جديدة لتصور الجيل سفينة من الخلايا الجذعية البطانية الأوعية الدموية

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

تصوير لايف من ماوس الثانوي باليت فيوجن

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

تشريح وتلطيخ إيممونوفلوريسسينت للفطر الجسم وعلم الخلايا العصبية في أدمغة البالغين melanogaster المورفولوجية

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

استخدام الموجات فوق الصوتية عالية التردد ثلاثي الأبعاد رواية لتوصيف الحمل المبكر في الماوس

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

زراعة أنسجة المبيض تصور الظواهر في المبيض الماوس

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

تقييم البارامترات أعصاب Ultrastructural بعد في توصيل الرحم النامي الماوس المخ والحبل الشوكي

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

تحديد كثافة القناة الصفراوية في كبد الفأر

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

الصب الوعائي لرئتي فأرة ما بعد الولادة المبكرة للتصوير المقطعي المجهري

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...