We are grateful to the Roney Family Foundation for the support of this study. We acknowledge the contribution of Dr. M. Lucrecia Alvarez to the planning and execution of the experiments described in this work.

ملاحظة: يتم توضيح الخطوات المتبعة في عزل exosomes البولية باستخدام طريقة الترسيب تعديل في الشكل (1) وتشمل الخطوات الأولية إزالة الخلايا الميتة الكبيرة والحطام الخلية بواسطة الطرد المركزي. بيليه النهائي تم الحصول عليها من طاف جمعها من كل خطوة لاحقة يتوافق مع exosome، والذي يذوب في محلول العزلة لمزيد من استخراج البروتين، RNA، DNA و. 1. جمع البول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل جمع البول، وإعداد مثبط البروتياز الكوكتيل وفقا لتعليمات الشركة الصانعة وإضافة 3.125 مل من هذا الكوكتيل إلى وعاء معقم فارغة. الأنبوب الذي يحتوي على كوكتيل مثبط البروتياز ويمكن بعد ذلك يتم توزيعها على المتطوعين لجمع البول. ملاحظة: السبب لإضافة مثبط البروتياز هو تقليل تدهور البروتين في عينة من البول. على الرغم من أننا تستخدم خصيصا كوكتيل مثبط البروتياز منسيغما، يمكن أن تكون بديلا غيرها من مثبطات الأنزيم البروتيني المتاحة تجاريا. </li><li style=";text-align:right;direction:rtl"> جمع حوالي 10 مل من البول باطلة أولا. معالجة البول مباشرة بعد جمع دون تخزين أو التبريد. عزل exosomes البولية في نسختين باستخدام طريقة الترسيب المحورة؛ واحدة من التكرارات هو لDNA، RNA الكلي واستخراج البروتين، في حين أن الآخر هو لتقدير حجم الجسيمات exosome البولية. ملاحظة: يمكن جمع عينة البول 24 ساعة لexosome العزلة عن طريق تخزين عند 4 درجات مئوية. </li></ol> 2. إزالة الحطام الخلية وتام-هورسفال البروتين (THP) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نقل عينات البول إلى 15 مل أنابيب الطرد المركزي. الطرد المركزي 10 مل من البول في 17،000 x ج لمدة 10 دقيقة في 37 ° C للقضاء على الخلايا والحطام الخلية (الشكل 1). </li><li style=";text-align:right;direction:rtl"> باستخدام ماصة، ونقل طاف لأنبوب جديد لمزيد من المعالجة. Resuspend وبيليه في 500 ميكرولتر من حل العزلة (250 ملي السكروز، 10 ملي triethanolamine، ودرجة الحموضة 7.6)، يليه 10 دقيقة الحضانة مع DL-dithiothreitol (DTT: نهائي تركيز 200 ملغ / مل) عند 37 درجة مئوية. يضاف DTT أن تتحلل THP، أي يصيد exosomes، مما يؤدي إلى انخفاض العوائد: ملاحظة. احرص على ترك بيليه دون عائق أثناء نقل طاف، وتأكيد أن طاف هو واضح من بيليه. </li><li style=";text-align:right;direction:rtl"> عينات بيليه دوامة كل 2 دقيقة حتى بيليه يذوب تماما. ثم إضافة 500 ميكرولتر من حل العزلة إلى بيليه المنحل. </li><li style=";text-align:right;direction:rtl"> على الفور بعد إضافة حل العزلة إلى بيليه، الطرد المركزي في 17،000 x ج لمدة 10 دقيقة عند 37 درجة مئوية. الماصة وطاف مع الجمع بين طاف جمعها في وقت سابق. </li><li style=";text-align:right;direction:rtl"> Resuspend وبيليه في 50 ميكرولتر من حل العزلة لتحليل SDS-PAGE. ملاحظة: برنامج تلفزيوني أو TBS يمكن استخدامها بدلا من حل العزلة إلى resuspend الكرية. </li></ol> 3. عزل Exosome بيليه <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إلى 11 مل من supern جمعهاatant، إضافة 3.3 مل من ExoQuick-TC كاشف واحتضان عند 4 درجة مئوية لمدة لا تقل عن 12 ساعة. ملاحظة: نسبة حجم طاف لExoQuick-TC كاشف يمكن تعديلها لزيادة أو تقليل العائد exosomal. لاحظنا أن كميات أقل من كاشف أضاف تنتج غلة أصغر من exosomes. </li><li style=";text-align:right;direction:rtl"> وبعد الحضانة، الطرد المركزي المزيج عينة / ExoQuick-TC في 10،000 x ج لمدة 30 دقيقة في درجة حرارة 25 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> نقل طاف لأنبوب جديد لتحليل SDS-PAGE. </li><li style=";text-align:right;direction:rtl"> ويمكن تخزين بيليه exosome في -80 درجة مئوية لمدة DNA، RNA واستخراج البروتين وتقدير حجم الجسيمات exosome باستخدام CD9 ELISA 11. ملاحظة: يمكن تخزين بيليه exosome في -80 درجة مئوية لمدة 1 سنة مع تجميد محدود والذوبان. </li></ol> 4. كشف العلامات البيولوجية وتحليل Exosome بيليه <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتحليل المصب، استخراج DNA و RNA والبروتين من بيليه exosome باستخدام DNA / RNA / طقم العزلة البروتين وكي العزلة RNAر وفقا لتعليمات الشركة الصانعة. تحديد تركيزات باستخدام معمل NanoDrop من خلال قياس الامتصاصية في 260 و 280nm مع 2 ميكرولتر من العينة. </li><li style=";text-align:right;direction:rtl"> أداء الكمي في الوقت الحقيقي RT-PCR (QPCR) باستخدام فحوصات الرنا الميكروي TAQMAN (في حالتنا، كنا الإنسان مير 192- ومير 1207-5p محددة المقايسات) أو الاشعال ميرنا محددة. </li><li style=";text-align:right;direction:rtl"> لتحليل SDS-PAGE، استخدم 4-12٪ مكرر تريس هلام وعينات البروتين منفصلة من قبل الكهربائي واحدة الأبعاد. تصور المواد الهلامية استخدام عدة الفضة تلطيخ وفقا لتعليمات الشركة الصانعة. </li><li style=";text-align:right;direction:rtl"> إجراء تحليل لطخة غربية باستخدام الأغشية PVDF وفقا للتعليمات المقدمة من قبل الشركة المصنعة. استخدام الأرنب بولكلونل الأجسام المضادة لموت الخلايا المبرمج الجين المرتبط-2 التفاعل بروتين X أو ALIX والفأر وحيدة النسيلة الأجسام المضادة للورم قابلية الجينات 101 البروتين أو TSG101 في الكشف طخة الغربي. قياس كثافة العصابات التي كتبها الوصول المفتوح البرمجيات NIH يماغيج (http://rsbweb.nih.gov/ij/) أوبرنامج مماثل. </li><li style=";text-align:right;direction:rtl"> تحديد عدد من الجزيئات exosome البولية تم الحصول عليها باستخدام مجموعة CD9 ELISA وفقا لتعليمات الشركة الصانعة. </li></ol>

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تم دفع رسوم النشر لهذا المقال من قبل العلوم البيولوجية الفرعية للتنفيذ.

معظم الطرق التي تنطوي على ترسيب exosomes من البول لا تعالج إزالة شبكة البوليمرية التي شكلتها البروتين تام-هورسفال (THP). هذا البروتين موجود في كميات كبيرة في البول، حيث exosomes أنه الفخاخ مزعومة أثناء الأولي الطرد المركزي بسرعة منخفضة. في أسلوبنا تعديل خفضنا THP باستخدام DTT قبل...

exosomes البولية هي الحويصلات الداخلية صغيرة من 100 نيوتن متر من الهيئات عديد الحويصلات (MVB) المستمدة من جميع أنواع الخلايا تحت الظروف العادية والمرضية في الفضاء البولية بما في ذلك podocytes الكبيبي، وخلايا النبيبات الكلوية والخلايا المبطنة للشبكات الصرف البولية التي يبدو أنها المخصب لmiRNAs 1 -2. وقد كشفت العديد من الباحثين البروتينات متميزة في exosomes معزولة عن البول الأفراد الأصحاء وكذلك مع كلوي و / أو الأمراض منهجية 3-5. Exosomes يمكن عزلها باستخدام طرق مختلفة مثل خطوتين التفاضلي تنبيذ فائق مع أو بدون السكروز 6-7، والجمع بين مرشح nanomembrane مع تنبيذ فائق 8-9، أو هطول الأمطار 10-11. لقد وضعت مؤخرا طريقة بسيطة وسريعة وقابلة للتطوير وفعال لعزل exosomes البولية وكشف ميرنا ومؤشرات حيوية البروتين 11. على الرغم من أن المؤشرات الحيوية يمكن اكتشافه في مجموعة متنوعة من السوائل البيولوجية المختلفة، اورالمعهد الوطني للإحصاء هو الخيار الأكثر ملاءمة للمرضى الذين يعانون أمراض الكلى والمسالك البولية لأنه يمكن الحصول على كميات كبيرة بطريقة نسبيا غير الغازية. على الرغم من أن هناك عدة طرق لعزل exosomes البولية 6-11، فإن الإجراء الأكثر شيوعا يعتمد على تنبيذ فائق التفاضلي مع وسادة السكروز 30٪. في حين أن هذا الأسلوب هو كفاءة وجودة exosomes الناتجة معزولة مع هذا الإجراء عالية، وأسلوب في حد ذاته يتطلب الموظفين المهرة وهي، والتي تتطلب العديد من الخطوات تنبيذ فائق تستغرق وقتا طويلا. أساليب أخرى، مثل الترشيح وهطول الأمطار، وقد وضعت لعزل exosomes، بما في ذلك واحد يستخدم الملكية هطول الأمطار كاشف (أي ExoQuick-TC: نظام العلوم البيولوجية، ماونتن فيو، كاليفورنيا). وعلى الرغم من هطول الأمطار باستخدام هذا كاشف بشكل سريع وسهل نسبيا، ونقاء exosomes معزولة منخفض بالمقارنة مع المنهجيات تنبيذ فائقالتطوير التنظيمي. ونحن مؤخرا مقابل ستة طرق لعزل exosomes البولية، بما في ذلك تعديل طريقة الترسيب باستخدام ExoQuick-TC التي قمنا بتطويرها في مختبرنا 11. وجدنا أن هذه الأمطار طريقة تعديل أثمرت كميات أكبر من البروتين، ميرنا، ومن mRNAs والإجراء نفسه كان أسرع وأسهل للتنفيذ من تنبيذ فائق. هنا، نحن تصف بروتوكول تجريبي لدينا هطول طريقة تعديلها بطريقة تدريجية، وتشمل مناقشة مزايا وعيوب هذه التقنية مقارنة مع غيرها من أساليب exosome العزلة. ويشمل هطول الأمطار على طريقة تعديل الأولي من الجسيمات exosome، تليها إزالة البروتين تام-هورسفال، الذي يرتبط مع بروتينات exosomal، والمعروف للحد من العائد exosome من البول. وجدنا أن هذه التعديلات زيادة المحصول ونقاء إعداد exosomal من البول.

<table border="0" cellpadding="0" cellspacing="0" width="736">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 15ml Centrifuge Tube</td>
			<td style="width:111px;">Corning (Sigma Aldrich)</td>
			<td style="width:119px;">CLS430791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease Inhibitor Cocktail</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:111px;">Sorvall</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-dithiothreitol</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">D9779</td>
			<td>used at final concentration of 200 mg/ml</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Novex NuPAGE SDS-PAGE system (4-12 % Bis-Tris Gel 1.0 mm, 10 well</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">NP0321BOX</td>
			<td>For SDS-PAGE analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ECL Advance Western blotting detection kit</td>
			<td style="width:111px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2135</td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exoquick-TC Reagent</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOTC50A-1</td>
			<td>For exosome isolation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exosome CD9 ELISA Complete Kit</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOEL-CD9-A-1</td>
			<td>For exosome quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AllPrep DNA/RNA/Protein mini kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">80004</td>
			<td>For DNA, RNA, Protein isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rneasy MinElute Cleanup kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">74204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>For Protein Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProteoSilver Sliver Stain Kit</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">PROTSIL1-1KT</td>
			<td>For staining SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xcell SureLock Mini-Cell Electrophoresis System</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For running the SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan microRNA assays</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For RT-PCR assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qBasePlus v1.5 software</td>
			<td style="width:111px;">Biogazelle NV</td>
			<td style="width:119px;"></td>
			<td>For analysis of RT-PCR assay data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit polyclonal antibody to ALIX</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse monoclonal antibody to TSG101</td>
			<td style="width:111px;">Abcam</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
	</tbody>
</table>

a modified precipitation method to isolate urinary exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

exosomes البولية تحمل البروتين، ميرنا ومرنا المؤشرات الحيوية يفرز من خلايا الظهارية الكلوية. عزل وكشف عن هذه exosomes قد توفر أداة تشخيصية مفيدة للكشف المبكر عن أمراض الكلى مثل اعتلال الكلية السكري. هناك عدة طرق لعزل exosomes من عينات البول التي جمعت من البشر. نحن سابقا مقابل ست?...

Watch this Scientific Journal Video about A Modified Precipitation Method to Isolate Urinary Exosomes at JoVE.com

A Modified Precipitation Method to Isolate Urinary Exosomes

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

وهناك طريقة الهطول معدلة لعزل البولية Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

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JoVE Journal

Medicine

17.5K Views.  Translational Genomics Research Institute (TGen). This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Video: A Modified Precipitation Method to Isolate Urinary Exosomes

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

طريقة حساسة لقياس خلايا السرطان هرمة

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

الفئران عنق الرحم القلب زرع نموذج باستخدام أزرار وتقنيات التعديل

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

وهناك طريقة معدلة لغيري مكاني ماوس القلب Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

المثانة العضلات الملساء قطاع انقباض كوسيلة لتقييم المسالك البولية السفلى الصيدلة

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

الهيكل العظمي إجراءات خزعة العضلات البشرية باستخدام تقنية التعديل بيرجستروم

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

تجربة سريرية لتقييم سلامة وفعالية وتسليم أكياس ثلاث غرف من زيت الزيتون للتغذية بالحقن

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

طريقة سيفينج فعالة لعزل Glomeruli سليمة من الفئران الكبار الكلي

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

التصوير بالموجات فوق الصوتية للمسالك البولية الذكور الكبار للاختبار الوظيفي البولي

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

طريقة بسيطة معدلة لتحريض احتشاء عضلة القلب في الفئران

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

A Modified Vessel-Sparing Microsurgical Vasoepididymostomy

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, preserving the vasal vessels during MVE might better simulate the normal physiological structure and be meaningful for patients who have undergone varicocelectomy. Nevertheless, preserving the vasal vessels might elevate the risk of increasing the tension of anastomosis, affecting the patency rate and leading to delayed postoperative bleeding. Therefore, we developed a novel vessel-sparing MVE to make it safer. Here is a summary of the improvements to the procedure. 1) The retrograde dissociation of the vasal vessels on the proximal testicular side was adopted as the main method, and the anterograde dissociation of the vasal vessels on the distal testicular side was adopted as a supplement to dissociate the vasal vessels to be preserved. This improvement ensures the blood supply to the vas deferens that will be used for anastomosis and also provides longer vasal vessels, which reduces the tension of anastomosis. 2) By fixing the vas deferens to be anastomosed and the broken end of the vas deferens, the free vasal vessels get fixed, which resolves the problem of transmission of vas tension to the vasal vessels and reduces the risk of vasal vessel hemorrhage. 3) Dissociation of the vas deferens after opening the tunica vaginalis increases the mobilization of the vas deferens, which also makes the new procedure easier to complete. The evaluation of the outcomes of this new procedure showed that no significant postoperative complications occurred in the patients, and the patency rate was no different from that of the conventional procedure. Therefore, this new, improved procedure can be considered safe, with satisfactory postoperative results.

Here, we present a protocol to preserve the vasal vessels in microsurgical vasoepididymostomy. The surgical security is enhanced by preserving the vasal vessels using a retrograde-anterograde dissociation and fixing the vasal vessels.

فغر الأوعية الدموية المجهري المعدل مع تجنيب الأوعية الدموية

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, ...