المؤلفون تقر ريان شابلن والدكتور جريج Rentfrow من جامعة كنتاكي اللحوم مختبر وقسم علوم الأغذية الحيوانية وتوفير فرص لجمع الأنسجة التجريبية المستخدمة في هذه الوثيقة.

<ol>
	<li>Matheson, P. J., Wilson, M. A., Garrison, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intestinal+blood+flow.">Regulation of intestinal blood flow.</a> The Journal of surgical research. 93, 182-196 (2000).</li><li>Dobson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+and+absorption+from+the+rumen.">Blood flow and absorption from the rumen.</a> Quarterly journal of experimental physiology. 69, 599-606 (1984).</li><li>Mulvany, M. J., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+vascular+smooth+muscle+cells+in+situ.">Mechanical properties of vascular smooth muscle cells in situ.</a> Nature. 260, 617-619 (1976).</li><li>Nielsen-Kudsk, F., Poulsen, B., Ryom, C., Nielsen-Kudsk, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strain-gauge+myograph+for+isometric+measurements+of+tension+in+isolated+small+blood+vessels+and+other+muscle+preparations.">A strain-gauge myograph for isometric measurements of tension in isolated small blood vessels and other muscle preparations.</a> Journal of pharmacological. 16, 215-225 (1986).</li><li>Mulvany, M. J., Aalkjaer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+small+arteries.">Structure and function of small arteries.</a> Physiological reviews. 70, 921-961 (1990).</li><li>Klotz, J. 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=Assessment+of+vasoconstrictive+potential+of+D-lysergic+acid+using+an+isolated+bovine+lateral+saphenous+vein+bioassay.">Assessment of vasoconstrictive potential of D-lysergic acid using an isolated bovine lateral saphenous vein bioassay.</a> Journal of animal science. 84, 3167-3175 (2006).</li><li>Klotz, J. L., Bush, L. P., Strickland, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vascular+contractility+bioassay+using+bovine+right+ruminal+artery+and+vein.">A vascular contractility bioassay using bovine right ruminal artery and vein.</a> Journal of animal science. 89, 1944-1951 (2011).</li><li>Westendorf, M. 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=In+vitro+and+in+vivo+ruminal+and+physiological+responses+to+endophyte-infected+tall+fescue.">In vitro and in vivo ruminal and physiological responses to endophyte-infected tall fescue.</a> Journal of dairy science. 76, 555-563 (1993).</li><li>Strickland, J. R., 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=Board-invited+review:+St.+Anthony&amp;#34;s+Fire+in+livestock:+causes,+mechanisms,+and+potential+solutions.">Board-invited review: St. Anthony&amp;#34;s Fire in livestock: causes, mechanisms, and potential solutions.</a> Journal of animal science. 89, 1603-1626 (2011).</li><li>Eckert, H., Kiechel, J. R., Rosenthaler, J., Schmidt, R., Schreier, E., B, B. e. r. d. e., HO, S. c. h. i. l. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+11.">Ch. 11.</a> Ergot Alkaloids and Related Compounds. , (1978).</li><li>Rhodes, M. T., Paterson, J. A., Kerley, M. S., Garner, H. E., Laughlin, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+blood+flow+to+peripheral+and+core+body+tissues+in+sheep+and+cattle+induced+by+endophyte-infected+tall+fescue.">Reduced blood flow to peripheral and core body tissues in sheep and cattle induced by endophyte-infected tall fescue.</a> Journal of animal science. 69, 2033-2043 (1991).</li><li>Foote, A. P., Harmon, D. L., Strickland, J. R., Bush, L. P., Klotz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ergot+alkaloids+on+contractility+of+bovine+right+ruminal+artery+and+vein.">Effect of ergot alkaloids on contractility of bovine right ruminal artery and vein.</a> Journal of animal science. 89, 2944-2949 (2011).</li><li>Foote, A. P., 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=Ergot+alkaloids+from+endophyte-infected+tall+fescue+decrease+reticuloruminal+epithelial+blood+flow+and+volatile+fatty+acid+absorption+from+the+washed+reticulorumen.">Ergot alkaloids from endophyte-infected tall fescue decrease reticuloruminal epithelial blood flow and volatile fatty acid absorption from the washed reticulorumen.</a> Journal of animal science. 91, 5366-5378 (2013).</li><li>Hocking, K. M., 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=Detrimental+effects+of+mechanical+stretch+on+smooth+muscle+function+in+saphenous+veins.">Detrimental effects of mechanical stretch on smooth muscle function in saphenous veins.</a> Journal of vascular surgery. 53, 454-460 (2011).</li><li>Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+intestinal+surgery.">Bovine intestinal surgery.</a> Modern veterinary practice. 65, 705-710 (1984).</li><li>Budras, K. D., Habel, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bovine Anatomy An Illustrated Text. , (2003).</li><li>Klotz, J. 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=Antagonism+of+lateral+saphenous+vein+serotonin+receptors+from+steers+grazing+endophyte-free,+wild-type,+or+novel+endophyte-infected+tall+fescue.">Antagonism of lateral saphenous vein serotonin receptors from steers grazing endophyte-free, wild-type, or novel endophyte-infected tall fescue.</a> Journal of animal science. 91, 4492-4500 (2013).</li><li>Egert, A. M., Kim, D. H., Schrick, F. N., Harmon, D. L., Klotz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+exposure+to+ergot+alkaloids+decreases+contractility+of+bovine+mesenteric+vasculature.">Dietary exposure to ergot alkaloids decreases contractility of bovine mesenteric vasculature.</a> Journal of animal science. 92, 1768-1779 (2014).</li></ol>

لا تشكل ذكر الاسم التجاري، والمنتج الملكية، أو المعدات المحددة كفالة أو ضمان من قبل وزارة الزراعة ولا يعني الموافقة على استبعاد غيرها من المنتجات التي قد تكون متاحة.

كان التحدي الأولي في تطوير هذا الأحيائي وإنشاء موقع لجمع تكرار الأوعية الدموية المساريقي. عينة الموقع الاتساق أمر بالغ الأهمية، حيث أن بعض مهام التغيير الأمعاء الدقيقة خلال التقدم من الصائم من خلال الدقاق وبالتالي مساريق تختلف في نمط مماثل. كانت فروع الشريان المسار...

التغيرات في تدفق الدم إلى الأنسجة سرير يمكن أن يكون لها تأثير كبير على وظيفة الجهاز. والوظيفة الرئيسية من الأمعاء الدقيقة لامتصاص العناصر الغذائية. مطلوب تدفق الدم الشرياني إلى السطح الاستيعابية للامعاء لامتصاص تدفق الدم وزيادة المغذيات للمساعدة في امتصاص العناصر الغذائية كما هضامة التحركات على طول السطح 1. وحدث انخفاض في تدفق الدم يمكن أن يسبب انخفاض في امتصاص العناصر الغذائية بسبب انخفاض في الانحدار بطريق الظهارة 2. بالإضافة إلى المواد الغذائية، ويمكن أيضا أن يتعرض الأمعاء الدقيقة إلى نواتج الأيض الثانوية، والمخدرات، أو السموم التي تمارس تأثير على تدفق الدم الموضعي في مساريق. في حالة الحيوانات المجترة، والمركبات يمكن ان تتحرر من الاعلاف (مثل المواد الغذائية مثل الأحماض الأمينية، أو السموم مثل قلويدات الشقران) من خلال عمليات التخمر من المعى الأمامي. إذا كانت هذه المركبات البقاء على قيد الحياة الأيض الميكروبي التخمير الكرش، فهي الآن متاحة للامتصاصأو التفاعل أثناء سفرهم من خلال القناة الهضمية للحيوان. هناك عدد من الأساليب المختلفة المتاحة لقياس تدفق الدم في الجسم الحي (على سبيل المثال، دوبلر بالموجات فوق الصوتية، ساكن مقاييس التدفق الدم، رديولبلد المجهرية، وتقنيات مؤشر-تمييع) التي تسمح تقييم السيناريوهات المختلفة أو العلاجات التجريبية. ومع ذلك، للحصول على معلومات حول الخصائص الميكانيكية أو الدوائية من العضلات الملساء الوعائية، وظلت أساليب محدودة للسفن الكبيرة حتى Mulvany وهالبيرن 3 نشرت مقالا يصف التقنية باستخدام سلك شنت الاستعدادات حلقة الأوعية الدموية في مخطاط العضل. منذ تطوير هذه التقنية ما زالت التعديلات التي ينبغي إدخالها على النظم المرتبطة مخطاط العضل التي تسمح مجموعة متنوعة من التطبيقات المختلفة لتقييم الهياكل الأنبوبية. كما تم تكييف هذا النظام لاستخدام قضبان ثابتة لتركيب السفن الكبيرة 4 حيث نضحلم يتم المطلوب التقنيات. بسبب الاختلاف في السفن من أصول التشريحية المختلفة والفروق في نفس السفن من أنواع مختلفة من الحيوانات، والبيانات من السفينة ونوع الحيوان لا يمكن بسهولة استقراء عبر السفن المختلفة أو نفس السفينة في أنواع حيوانية مختلفة 5. وبالتالي، يجب تطوير اختبارات بيولوجية منفصلة والتحقق من صحة أي وقت يتم تغيير هذه الجوانب. في الآونة الأخيرة تم تطوير عدة اختبارات بيولوجية مع هذه التقنيات لاستخدامها في الماشية الوحشي الوريد الصافن والشريان والوريد الأيمن الكرش 6،7. وقد وضعت هذه الأحيائي للتحقيق على وجه التحديد الآثار التي قلويدات الشقران لها على دعم الأوعية الدموية في الأمعاء الدقيقة. أفيد بأن 50-60٪ من القلويدات تغذية تظهر في محتويات ابوماسال، ولكن يتم استرداد 5٪ فقط في البراز 8. وذكر ستريكلاند وآخرون. 9 في مراجعة قلويدات الشقران، التي سوج البيانات المتاحةطبقا لتقديرات أن الأمعاء الدقيقة قد تكون أهم موقع لامتصاص ergopeptine. إيكرت وآخرون 10 الجوانب الصيدلانية البيولوجية استعرض قلويدات من الشقران وذكرت أنه بمجرد عبورهم الحاجز الظهاري، ويتم نقل قلويدات الشقران إما عن طريق الجهاز اللمفاوي إلى الوريد تحت الترقوة أو عن طريق الوريد المساريقي والى الدم البابي. رودس وآخرون 11 سجلت انخفاضا في تدفق الدم إلى الاثني عشر والقولون في يرسم تستهلك عالية المصابة نابوت داخلي (عالية الشقران قلويد) النظام الغذائي. باستخدام الصحيح الكرش الشريان والوريد الأحيائي، فوت وآخرون 12 أثبتت أن قلويدات الشقران هي فعال في الأوعية في الأوعية الدموية الكرش. فوت وآخرون 13 أظهرت في وقت لاحق في الجسم الحي أن التعرض الكرش إلى نتائج قلويدات الشقران في الكرش انخفاض تدفق الدم الظهارية. هذا الانخفاض في تدفق الدم إلى السطح الاستيعابية للالكرش تسبب بالتزامن انخفاض في المغذيات (الأحماض الدهنية المتطايرة) تدفق. نظرا لتشوantity من قلويدات الشقران يمر إلى الأمعاء الدقيقة من المعى الأمامي. كان الافتراض بأن تأثير مماثل على الأوعية الدموية في الأمعاء الصغيرة وامتصاص العناصر الغذائية يمكن أن يحدث. هذا استلزم تطوير البقري القريبة الشريان المساريقي اللفائفي والوريد الأحيائي.

<table border="0" cellpadding="0" cellspacing="0" width="611">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:105px;">Name of the Material/Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:259px;">Comment/Description (optional)</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:105px;">Multi Myograph</td>
			<td style="width:127px;">Danish Myo Technologies</td>
			<td style="width:120px;">610M</td>
			<td style="width:259px;">A myograph is critical to this bioassay, but there are other platforms available for use that will suffice.&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">Powerlab 8/sp</td>
			<td style="width:127px;">ADIntruments</td>
			<td style="width:120px;">ML785</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">LabChart 7</td>
			<td style="width:127px;">ADInstruments</td>
			<td style="width:120px;">Version 7</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Force Calibration Kit</td>
			<td style="width:127px;">Danish Myo Technologies</td>
			<td style="width:120px;">100055</td>
			<td style="width:259px;">Specific to DMT myographs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Bottle-top Filter</td>
			<td style="width:127px;">Nalgene</td>
			<td style="width:120px;">595-4520</td>
			<td style="width:259px;">0.22 um pore size; 45 mm neck size</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">#5 Jewler&#8217;s Forceps</td>
			<td style="width:127px;">Miltex</td>
			<td style="width:120px;">555008FT</td>
			<td style="width:259px;">Any brand of forceps can be used</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Noyes Iris Scissors</td>
			<td style="width:127px;">Miltex</td>
			<td style="width:120px;">18-1510</td>
			<td style="width:259px;">Any brand of scissors can be used</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Dissecting Scope</td>
			<td style="width:127px;">Zeiss</td>
			<td style="width:120px;">Stemi 2000-C</td>
			<td style="width:259px;">Any brand of dissecting light microscope will suffice</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:105px;">Adjustable Tissue Matrice</td>
			<td style="width:127px;">Braintree Scientific</td>
			<td style="width:120px;">TM C12</td>
			<td style="width:259px;">This is not critical to the assay, but greatly reduces section to section variation in length and speeds up the slicing process greatly</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Krebs-Hensleit Buffer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">K3753-10x1L</td>
			<td style="width:259px;">It is not necessary to buy Krebs, this can be made in house</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:105px;">Calcium chloride dehydrate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">C7902-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Sodium bicarbonate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">S5761-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Desipramine-HCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">D3900-5G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Propranolol-HCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">P0884-1G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">KCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">P9333-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:105px;">95% O2/5% CO2</td>
			<td style="width:127px;">Scott Gross</td>
			<td style="width:120px;">UN3156</td>
			<td style="width:259px;"></td>
		</tr>
	</tbody>
</table>

isolating and using sections of bovine mesenteric artery and vein as a bioassay to test for vasoactivity in the small intestine

يتعرض الجهاز الهضمي الثدييات باستمرار لمركبات (المرغوبة وغير المرغوبة) التي يمكن أن يكون لها تأثير على تدفق الدم من وإلى هذا النظام. التغيرات في تدفق الدم إلى الأمعاء الدقيقة يمكن أن يؤدي إلى آثار على وظائف الاستيعابية للجهاز. وقد وضعت مصلحة خاصة في السموم تتحرر من الأعلاف من خلال عمليات التخمر والهضم في المجترات كمجال حيث يمكن تحسين الكفاءة الإنتاجية. الفيديو المرتبطة بهذا المقال يصف الأحيائي في المختبر وضعت لمركبات الشاشة لvasoactivity معزولة في المقاطع العرضية من الشريان المساريقي البقري والوريد باستخدام multimyograph. مرة واحدة هي التي شنت الأوعية الدموية ومعايرتها في مخطاط العضل، والأحيائي نفسها يمكن استخدامها: كأداة فحص لتقييم الاستجابة مقلص أو vasoactivity من المركبات المصالح؛ تحديد وجود أنواع مستقبلات خلال استهداف دواء مستقبلات مع ناهض محددق. تحديد دور مستقبلات مع وجود واحد أو أكثر من الخصوم. أو تحديد التفاعلات المحتملة للمركبات في المصالح مع الخصوم. من خلال كل هذا، يتم جمع البيانات في الوقت الحقيقي، والأنسجة التي تم جمعها من حيوان واحد يمكن أن يتعرض لعدد كبير من العلاجات التجريبية المختلفة (ميزة في المختبر)، ويمثل الأوعية الدموية على جانبي السرير الشعرية لتوفير دقة صورة لما يمكن أن يحدث في وارد وصادر دعم إمدادات الدم إلى الأمعاء الدقيقة.

تم جمع الأوعية الدموية تستخدم لتوليد النتائج المتضمنة في الفترة من 6 يرسم هولشتاين (425 ± 8 كلغ) ضمن فترة 3 أسابيع. ويرد مثال على استجابة الوريد المساريقي نموذجي مقلص لبوكل والإضافات العلاج زيادة في التركيز في الشكل 2. سيكون حجم الاستجابة تختلف بعض مع حجم السفين?...

Watch this Scientific Journal Video about Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine at JoVE.com

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

وكثيرا ما تتعرض الأمعاء الدقيقة للسموم التي يمكن أن تؤثر على تدفق الدم وتؤثر سلبا على امتصاص العناصر الغذائية. باستخدام الشريان المساريقي وmultimyograph ويعزل الوريد، المركبات أو السموم من الفائدة يمكن فرزهم لvasoactivity.

عزل وعن طريق أقسام الأبقار الشريان والوريد المساريقي باعتباره الأحيائي لاختبار لVasoactivity في الأمعاء الدقيقة

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

isolating-using-sections-bovine-mesenteric-artery-vein-as-bioassay-to

Research

JoVE Journal

Medicine

9.6K Views.  USDA-Agricultural Research Service.  وكثيرا ما تتعرض الأمعاء الدقيقة للسموم التي يمكن أن تؤثر على تدفق الدم وتؤثر سلبا على امتصاص العناصر الغذائية. باستخدام الشريان المساريقي وmultimyograph ويعزل الوريد، المركبات أو السموم من الفائدة يمكن فرزهم لvasoactivity. 

Video: Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

استراتيجية لتحديد الطفرات دي نوفو في الاضطرابات الشائعة مثل التوحد والفصام

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

الشريان المساريقي تقلص والاسترخاء عن طريق الدراسات الآلي Myography أسلاك

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

الكبار ماوس وريدي ارتفاع ضغط الدم نموذج: الشريان السباتي المشترك لجهات خارجية الوداجي الوريد التحام.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

تقييم استجابة عضلي وVasoactivity في الشرايين المقاومة مساريقي عن طريق تخطيط العضل الضغط

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

القسطرة من الشريان السباتي والوريد الوداجي إلى تنفيذ تدابير الدورة الدموية، الحقن والدم أخذ العينات في وإدراكا نموذج الفأر

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

الحقن الشريان السباتي لوأضافا الدوائية وتحليل Taxanes في الفئران

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

جهاز بسيط للتحضير السريع الجبال الجامعة للماوس الأمعاء

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

متساوي القياس كونتراكتيليتي القياس الشريان المساريقي العلوي الماوس باستخدام أسلاك ميوجرافي

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

الآليات الأساسية لإفراز هرمون الأمعاء التي تستخدم عزل Perfused الفئران الأمعاء

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

جسيمات نانوية المغلفة بولييثيلينيميني أكسيد الحديد كوسيلة لإيصال الحمض النووي الريبي التدخل الصغيرة إلى الضامة في المختبر و في فيفو

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...