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.

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.2K 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

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

Spinotrapezius نموذج الفئران لتقييم أثر ربط شريني ظيفة الاوعية الدموية الدقيقة وعلى إعادة عرض

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

Medicine

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 ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

القلب والأوعية الدموية التصوير باستخدام نظام الموجات فوق الصوتية عالية التردد (MHZ 30/45) الجنينية الماوس

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

الموجات فوق الصوتية عالية التردد لتحليل وضع الجنين والمشيمة في فيفو

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

هل فأري حامل؟ تقييم الموجات فوق الصوتية عالية التردد

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

طرق دراسة مساهمات الرحم في تأسيس الحمل في نموذج فأر استئصال المبيض

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

نموذج الفئران من هيبوبيرفوسيون داخل الرحم خفيفة مع تضيق ميكروكويل

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Neuroscience

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

تخطيط الصدى في تحقيقات الإنجابية تجريبية على الفئران

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

القياس الكمي للمعلمات الوعائية في شبكية العين الكاملة للفئران ذات اعتلال الشبكية غير التكاثري والتكاثري

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Development of a Murine Model for Femoral Artery Anastomotic Stenosis

In vascular surgery, vascular anastomosis is a common reconstructive technique used to restore blood flow. However, anastomotic restenosis is a frequent postoperative complication, primarily caused by surgery-induced vascular injury, intimal hyperplasia, and inflammatory responses. The mouse femoral artery anastomosis model is widely used to investigate the mechanisms of anastomotic restenosis and vascular repair. Microscopically guided end-to-end femoral artery anastomosis allows precise simulation of vascular injury and repair processes following surgery, providing a reliable experimental tool for studying the pathological mechanisms related to restenosis. This study focuses on refining the surgical technique for femoral artery anastomosis in mice. Through refinements in surgical techniques and optimization of technical details, we have achieved a marked increase in the success rate and reproducibility of the model. Specific improvements include refined vascular handling techniques during surgery, the selection of suture materials, and the optimization of suturing methods to minimize anastomotic leakage and postoperative occlusion. The study also emphasizes the observation of intimal hyperplasia, vascular remodeling at the anastomotic site, and long-term vessel patency. Through this research, we provide a concise and efficient operational guide for performing mouse femoral artery anastomosis, offering reliable technical support for experimental studies in vascular surgery. This work lays a solid foundation for subsequent investigations into related mechanisms and evaluations of therapeutic intervention.

Here we present a murine model for femoral artery anastomosis, offering researchers a valuable animal model to study and simulate vascular anastomotic stenosis. This development is crucial for advancing our understanding of the pathophysiology underlying this condition and facilitating more accurate and effective research into vascular diseases.

In vascular surgery, vascular anastomosis is a common reconstructive technique used to restore blood flow. However, anastomotic restenosis is a frequent ...