نشكر كريستا دن Ouden لها المساعدة التقنية الممتازة. وأيد هذه التقنية ماليا من قبل مؤسسة الكلى الهولندية، ومنحة C06.2174. ويدعم MCV من قبل المنظمة الهولندية للأبحاث العلمية (NWO) VIDI-منحة 016.096.359.

يتم تنفيذ جميع التجارب وفقا لدليل تجريبي خطوط الأخلاقية الحيوانية للجنة الحيوان أوتريخت التجريبية. يتم تنفيذ البروتوكول بتوجيه وموافقة من مؤسسة رعاية الحيوان المؤلف واللجنة الاستخدام. هو فعل كد في ذكر الفطرية لويس الفئران (تشارلز ريفر، Sulzfeld، ألمانيا) في سن 8 أسابيع. يتم إيواء الفئران تحت الظروف القياسية في بيئة، وعلى ضوء درجة الحرارة والرطوبة التي تسيطر عليها. 1. التحضير لعملية جراحية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعقيم الأدوات الجراحية: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 1 الأنسجة طالب ملقط 1-2 أسنان 12 سم </li><li style=";text-align:right;direction:rtl"> 1 طالب ملقط نمط القياسية </li><li style=";text-align:right;direction:rtl"> 2 ملقط Semken </li><li style=";text-align:right;direction:rtl"> 1 مايو مقص </li><li style=";text-align:right;direction:rtl"> 1 طالب مقص القزحية </li><li style=";text-align:right;direction:rtl"> 1 أولسن-هيغار حامل إبرة مع مقص </li><li style=";text-align:right;direction:rtl"> بطانية </li></ul></li><li style=";text-align:right;direction:rtl"> تحقق قائمة الجرد: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> طاولة العمليات مع لوحة الاحترار ومصباح </li><li style=";text-align:right;direction:rtl"> O <suب&gt; 2، isoflurane و </li><li style=";text-align:right;direction:rtl"> الأنسجة </li><li style=";text-align:right;direction:rtl"> صبي </li><li style=";text-align:right;direction:rtl"> موازين </li><li style=";text-align:right;direction:rtl"> مجموعة التشغيل العقيمة </li><li style=";text-align:right;direction:rtl"> 5X5 شاش معقم و10X10 </li><li style=";text-align:right;direction:rtl"> 70٪ كحول </li><li style=";text-align:right;direction:rtl"> 0.9٪ محلول كلوريد الصوديوم </li><li style=";text-align:right;direction:rtl"> 1 مل حقنة </li><li style=";text-align:right;direction:rtl"> الإبر (25G) </li><li style=";text-align:right;direction:rtl"> جل فوم: spongostan </li><li style=";text-align:right;direction:rtl"> VICRYL 4.0 و 5.0 </li><li style=";text-align:right;direction:rtl"> البوبرينورفين 0.03 مغ / كغ (1:10 المخفف في الملح الفسيولوجية) </li><li style=";text-align:right;direction:rtl"> قفص نظيفة للفئران بعد الجراحة </li></ul></li></ol> 2. استئصال إحدى الكليتين الجانب الأيمن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تطهير الجدول مع الكحول 70٪. </li><li style=";text-align:right;direction:rtl"> مكان الفئران في مربع الاستقراء ولحث التخدير مع 4٪ isoflurane وو 1 لتر من تدفق O 2. ملاحظة: اعتمادا على سلالة الفئران المستخدمة، توصف قبل وتسكين المحيطة بالجراحة، وهذا يحتاج إلى أن تناقش مع الطبيب البيطري المحلي. </li><li style=";text-align:right;direction:rtl"> نقل الفئران إلى طاولة ومكان الأنف في مخروط. يتم وضع الفئران ومخروط الأنف على وسادة التدفئة للحفاظ على درجة حرارة الجسم في الجرذ. الفئران أناق التهوية بمزيج من 2٪ isoflurane وو 1 ليتر O 2. </li><li style=";text-align:right;direction:rtl"> حلاقة الجهة من الفئران وتعقيم مع الكحول. غسل وتطهير اليدين وارتداء القفازات الجراحية المعقمة. ارتداء قبعة الرأس وقناع. جعل بنتيجة 1 - 1.5 سم شق بالتوازي مع الأضلاع باستخدام ملقط التشريحية ومقص حادة. فضح الكلى عن طريق تشريح حادة في عضلات الظهر. </li><li style=";text-align:right;direction:rtl"> القطب السفلي من الكلى مرئيا، بعناية استخدام الملقط حادة صغيرة لقبضة الأنسجة الدهنية محيط بالكلية. تخريج الكلى عن طريق سحب بلطف على الدهون محيط بالكلية مع ملقط. الحرص على عدم تعكير صفو الغدة الكظرية أثناء هذا الإجراء. الغدة الكظرية ويمكن إزالتها بسهولة عن طريق وضع ملقط في موقع وسطي في الأنسجة الدهنية وانتقلت بلطف صعودا بين الغدة الكظرية والكلى كما هو مبين في الشكل 2. </li><li style=";text-align:right;direction:rtl"> وضع الكلى بلطف على شاش واضحة من الأنسجة المحيطة الدهون والضامة. تحديد الشريان الكلوي والوريد ووضع رباط (5.0VICRYL) مع عقدة واحدة حول الأوعية، ولكن لا ربط قبالة عقدة. </li><li style=";text-align:right;direction:rtl"> تحريك عقدة فضفاضة بعناية على طول الأوعية نحو الشريان الأورطي من قبل ما يقرب من 0.5 سم، لخلق مساحة بين الكلى وعقدة. ويتم ذلك لمنع ضمد من الانزلاق بعد القطع. </li><li style=";text-align:right;direction:rtl"> ربط عقدة مع اثنين من عقدة مزدوجة. إذا توقفت التروية، فإن اللون في الكلى تغيير على الفور إلى البني. لا تقطع نهايات ضمد. </li><li style=";text-align:right;direction:rtl"> قطع الأوعية الكلوية على مقربة من الكلى وإزالة الكلى. سحب بلطف على الغايات طويلة من ضمد للتحقق من نزيف من الأوعية الكلوية. ربطة يجب أن يبقى في مكان على متن السفينة. قطع نهايات طويلة من ضمد. ملاحظة، سوف الأوعية الكلوية بقايا التراجع في تجويف البطن على الفور. </li><li style=";text-align:right;direction:rtl"> لطخة الكلى رفعه الجافة وتزن عليه. </li><li style=";text-align:right;direction:rtl"> إغلاق شق العضلات والهيكل العظمي مع تشغيل الغرز مع VICRYL 5.0. حقن 0.10 مل (0.03 ملغ / كلغ) بوبرينورفين ايم في أطرافه الخلفية. إغلاقالجلد مع VICRYL 4.0 مع الغرز داخل الجلد. هذا يمنع الفئران من فتح الجرح. ملاحظة: نظرا للاكتئاب الجهاز التنفسي في الفئران لويس، ونحن لا تعطي إلا تسكين بعد العملية فقط قبل إغلاق isoflurane و. </li><li style=";text-align:right;direction:rtl"> إيواء الفئران في قفص الانفرادي نظيفة مع سهولة الوصول إلى الطعام والماء. ضع نصف القفص على الاحترار سادة O / N والتحقق من الفئران بعناية في اليوم التالي. عندما يتم إغلاق الجرح والفئران نشطا، والذي هو عادة بين 6 و 12 ساعة بعد الجراحة، والفئران يمكن وضعها مرة أخرى إلى السكن المجموعة. </li></ol> 3. الجانب الأيسر استئصال الكلية المجموع الفرعي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد سبعة أيام، يتم إجراء نفس الاستعدادات على الجانب الأيسر أما الجانب الأيمن كما هو موضح 2،1-2،5. قبل الجراحة، يتم حساب كمية الأنسجة الكلوية التي تحتاج إلى إزالة المقابلة لحوالي 2/3rds من وزن الكلية اليمنى. إعداد قطع صغيرة من الرغوة هلام (Spongostan، وجونسون آند جونسون، نيو جيرسي) قبل الجراحة. </lI&gt; <li style=";text-align:right;direction:rtl"> ضع مقص حاد حول القطب العلوي من الكلية اليسرى وبتر القطب العلوي في ضربة واحدة (على موقع مقص، انظر الشكل 3). تغطية مباشرة مع قطعة من رغوة جل وممارسة ضغوط خفيفة مع شاش معقم. </li><li style=";text-align:right;direction:rtl"> كرر مع القطب السفلي من الكلى كما هو الحال في 3.3. </li><li style=";text-align:right;direction:rtl"> رفع الكلى لمنع تخثر الدم على الجلد. الحفاظ على ضغط معتدل على كلا منصات رغوة الجل مع شاش معقم حتى يتوقف النزيف. عندما استمر النزيف، إضافة وسادة رغوة آخر. </li><li style=";text-align:right;direction:rtl"> وضع بقايا الكلى مع ملتصقة فوم هلام داخل البطن. إغلاق العضلات والجلد واتبع 2،9-2،12. </li></ol> 4. الشام جراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضوابط تعمل الشام الخضوع لنفس الإجراء من أجل فضح الكلى. وبدلا من اقتلاع الكلى أو قطع القطبين بعد أسبوع واحد يتم الأرتيميا Decapsulated كلا الكلى في فترة أسبوع واحد، مع الحرص على عدم تعكير صفو الغدد الكظرية. إغلاق الجرح والرعاية اللاحقة للعمليات الجراحية هي معرفentical إلى 2.11 و 2.12. </li></ol>

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الاستئصال الجراحي لل5/6th من كتلة الكلوي في الفئران لويس، جنبا إلى جنب مع اتباع نظام غذائي غني بالملح وNOS تثبيط مؤقت يؤدي إلى نموذج من كد كد يشبه الإنسان وتسمح دراسة الآليات السببية ونجاعة التدخلات العلاجية في كد. نموذج استئصال الك?...

ونظرا لطبيعته التقدمية، التي تلت نهاية المرحلة مرض الكلى، والاعتلال القلب والأوعية الدموية المرتبطة بها والوفيات، كد هي مشكلة متنامية للصحة العامة 1. وبالتالي تباطؤ التقدم كد من الأولويات الصحية الرئيسية. منذ يتميز كد بواسطة خللا المعقدة من التوازن، النماذج الحيوانية تكاملية ضرورية لدراسة تطور وتقدم من كد. تتكون الكلى من مجموعة واسعة من أنواع مختلفة من الخلايا التي تتفاعل مع بعضها البعض. لا يمكن أن تحاكي هذا التعقيد في المختبر. لدراسة التدخلات العلاجية رواية في كد، ونحن نستخدم نموذج 5/6th استئصال الكلية الاجتثاث، وهو نموذج تجريبي معروفة من مرض الكلى التدريجي، تشبه عدة جوانب من كد الإنسان 2،3. ويصل إجمالي التخفيض في كتلة الكلوي الكبيبي يسبب التدريجي وإصابة انبوبية-الخلالي، وفقدان النيفرون بقايا والتنمية من ارتفاع ضغط الدم النظامية والكبيبي. يترافق ذلك مع البرامج الحديثة اضافة بالصورessive ضمن الكلية فقدان شعري 4، التهاب الكبيبات و. عوامل الخطر لكد دائما تأثير على وظيفة بطانة الأوعية الدموية 5. كنا سلالة الفئران (لويس) التي هي مقاومة للتطوير كد نسبيا، وبالتالي فإننا مجتمعة إزالة 5/6th من كتلة الكلوي مع أكسيد النيتريك (NO) استنفاد 6، 7، 8 واتباع نظام غذائي ملح عالية 9. بعد وصول والتأقلم، والحيوانات تحصل على NO المانع سينسيز (L-NNA) تستكمل لمياه الشرب (20 ملغم / لتر) لمدة 4 أسابيع، تليها استئصال إحدى الكليتين جانب الحق (UNX) مع استمرار L-NNA بعد يومين . بعد أسبوع واحد، يتم تنفيذ المجموع الفرعي استئصال الكلية (SNX) أي إزالة 2/3rds من كتلة الكلوي على الجانب الأيسر. بعد SNX، يسمح للحيوانات لاسترداد لمدة 2 أيام ثم مرة أخرى بنسبة 20 ملغ / لتر في مياه الشرب LNNA لمدة 4 أسابيع. واصلت اتباع نظام غذائي عالية من الملح (6٪)، على أن تستكمل في الأرض تشاو (انظر الإطار الزمني الشكل 1)، في كافة مراحل التجربة. Tانه سبب لأداء UNX على الجانب الأيمن وSNX على الجانب الأيسر هو أن الأوعية الكلوية هي أطول على الجانب الأيسر مما يجعل من الاسهل للوصول إلى الكلى دون تمتد الأوعية كثيرا عندما يتعرض الكلى خارج الجسم . في الأدب، وصفت النماذج التي يتم إزالة أعمدة من الكلية اليسرى أولا، تليها UNX من الكلية اليمنى بعد أسبوع واحد 10،11،12. في أيدينا أظهر هذا النموذج أكثر بكثير السريع تطوير الفشل الكلوي، ولكن أيضا تباين أكبر بكثير في فقدان وظائف الكلى. ويتبع تطور الفشل الكلوي مع مرور الوقت من خلال قياس البلازما اليوريا، وضغط الدم الانقباضي وبروتينية. من قبل ستة أسابيع بعد SNX، وقد وضعت الفشل الكلوي، والتي تتميز انخفاض ملحوظ في معدل الترشيح الكبيبي (69٪) وتدفق البلازما الكلوي فعالة (62٪) 13 ارتفاع ضغط الدم (ضغط الدم الانقباضي&gt; 150 مم زئبق)، بروتينية (&gt; 50 mg/24 HR) ومعتدل يوريمية (&gt; 10 ملم). وتشمل الميزات النسيجية tubulالضرر O-الخلالي التي تعكسها التهاب، ضمور أنبوبي وتليف وتصلب الكبيبات التنسيق مما أدى إلى انخفاض كبير من الكبيبات صحي بين السكان بقايا (&lt;10٪). المتابعة حتى بعد 12 أسبوعا تبين SNX مزيد من التقدم من كد، وتوفير فرصة لتقييم التدخلات العلاجية.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>L-NNA</td>
			<td>Sigma-aldrich</td>
			<td>N5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spongostan dental: gel foam pads 1x1x1 cm</td>
			<td>Johnson&amp;Johnson</td>
			<td>Ms0005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl FS-2S naald 4/0 V392H p/36</td>
			<td>Ethicon</td>
			<td>V303H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl RB-1+ naald 5/0 V303H p/36</td>
			<td>Ethicon</td>
			<td>V392H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine (0.3 mg/ml)</td>
			<td>Via local pharmacist ordered by Reckitt Benckiser pharmaceuticals</td>
			<td>unknown</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Student Tissue Forceps - 1x2 Teeth 12 cm</td>
			<td>Fine Science Tools (FST)</td>
			<td>91121-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forceps</td>
			<td>FST</td>
			<td>91100-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>FST</td>
			<td>14010-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2X Semken Forceps</td>
			<td>FST</td>
			<td>11008-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Iris Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>FST</td>
			<td>12002-14</td>
			<td>&nbsp;</td>
		</tr>
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5/6th nephrectomy in combination with high salt diet and nitric oxide synthase inhibition to induce chronic kidney disease in the lewis rat

مرض مزمن في الكلى (كد) هو مشكلة عالمية. تباطؤ التقدم كد من الأولويات الصحية الرئيسية. منذ يتميز كد بواسطة خللا المعقدة من التوازن، النماذج الحيوانية تكاملية ضرورية لدراسة تطور وتقدم من كد. لدراسة تطوير التدخلات العلاجية كد والرواية في كد، ونحن نستخدم نموذج الاجتثاث استئصال الكلية 5/6th، وهو نموذج تجريبي من المعروف جيدا من مرض الكلى التدريجي، تشبه عدة جوانب من كد الإنسان. ويصل إجمالي التخفيض في كتلة الكلوي الكبيبي يسبب التدريجي وإصابة انبوبية-الخلالي، وفقدان النيفرون بقايا والتنمية من ارتفاع ضغط الدم النظامية والكبيبي. كما انه يرتبط مع تقدمية ضمن الكلية الشعرية الخسارة، التهاب الكبيبات و. عوامل الخطر لكد دائما تأثير على وظيفة بطانة الأوعية الدموية. لتقليد هذا، ونحن الجمع بين إزالة 5/6th من كتلة الكلوي مع أكسيد النيتريك (NO) استنزاف واتباع نظام غذائي عالي الملح. بعد وصول والتأقلم، وإعادة الحيواناتالوقت المتفق عليه لNO المانع سينسيز (NG-نيترو-L-أرجينين) (L-NNA) تستكمل لمياه الشرب (20 ملغم / لتر) لمدة 4 أسابيع، تليها استئصال إحدى الكليتين جانب الحق. بعد أسبوع واحد، يتم إجراء استئصال الكلية المجموع الفرعي (SNX) على الجانب الأيسر. بعد SNX، يسمح للحيوانات لاسترداد لمدة يومين تليها LNNA في مياه الشرب (20 ملغم / لتر) لفترة أخرى مدتها 4 أسابيع. واصلت اتباع نظام غذائي عالية من الملح (6٪)، على أن تستكمل في الأرض تشاو (انظر الإطار الزمني الشكل 1)، في كافة مراحل التجربة. ويتبع تطور الفشل الكلوي مع مرور الوقت من خلال قياس البلازما اليوريا، وضغط الدم الانقباضي وبروتينية. من قبل ستة أسابيع بعد SNX، وقد وضعت الفشل الكلوي. يتم قياس وظائف الكلى باستخدام حبوب &quot;المعيار الذهبي&quot; وحمض الهيبوريك شبه الأمينية (PAH) تكنولوجيا إزالة الألغام. ويتميز هذا النموذج من كد من انخفاض في معدل الترشيح الكبيبي (GFR) وتدفق البلازما الكلوي الفعال (ERPF)، ارتفاع ضغط الدم (ضغط الدم الانقباضي&gt; 150 مم زئبق)، بروتينية (&gt; 50 mg/24 ساعة) ويوريمية معتدل (&gt; 10 ملم). وتشمل الميزات النسيجية الضرر انبوبية-الخلالي التي تعكسها التهاب، ضمور أنبوبي وتليف وتصلب الكبيبات التنسيق مما أدى إلى انخفاض كبير من الكبيبات صحي بين السكان بقايا (&lt;10٪). المتابعة حتى بعد 12 أسبوعا تبين SNX مزيد من التقدم من كد.

بعد استئصال الكلية المجموع الفرعي، يتم ترك حوالي 1/6th من مجموع كتلة الكلوي. ويبين الشكل 4 وزن الجزء إزالة من الكلية اليمنى مع المتوسط ​​والانحراف المعياري في اثنين من التجارب السابقة. ينبغي للمرء أن نضع في اعتبارنا أنه في الأسبوع بعد UNX، يحدث تضخم في الكلية ا?...

Watch this Scientific Journal Video about 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat at JoVE.com

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

ووصف طريقة مرحلتين لإنشاء مرض الكلى المزمن (كد) في الفئران لويس عن طريق إزالة جراحيا 5/6th من كتلة الكلوي. مزيج من إجراء العمليات الجراحية، NOS-تثبيط واتباع نظام غذائي غني بالملح يؤدي إلى نموذج يشبه كد الإنسان، والسماح دراسة الآليات السببية وتطوير تدخلات علاجية جديدة.

5/6th استئصال الكلية في تركيبة مع حمية عالية الملح والنيتريك أوكسيد تثبيط سينسيز للحث على مرض الكلى المزمن في الفئران لويس

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

56th-nephrectomy-combination-with-high-salt-diet-nitric-oxide

Research

JoVE Journal

Medicine

23.4K Views.  University Medical Center Utrecht. ووصف طريقة مرحلتين لإنشاء مرض الكلى المزمن (كد) في الفئران لويس عن طريق إزالة جراحيا 5/6th من كتلة الكلوي. مزيج من إجراء العمليات الجراحية، NOS-تثبيط واتباع نظام غذائي غني بالملح يؤدي إلى نموذج يشبه كد الإنسان، والسماح دراسة الآليات السببية وتطوير تدخلات علاجية جدي?...

Video: 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

التحليلية تقنيات يعاير النيتريك النشاط الحيوي أوكسيد

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

اجتثاث القثطرة في تركيبة مع الزمن لاختتام أطرافهم الأذيني الرجفان الأذيني

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

الطيفية Karyotyping إلى دراسة حالة شذوذ الكروموسومات في البشر والفئران مع مرض الكلى المتعدد الكيسات

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

نموذج من تسريب المغذيات المزمنة في الفئران

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

تقييم وظيفة الأوعية الدموية في المرضى الذين يعانون من مرض الكلى المزمن

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

بطء الإفراج تسليم المخدرات من خلال Elvax 40W إلى الجرذ الشبكية: الآثار المترتبة على علاج الحالات المزمنة

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

بوسلفان باعتباره وكيل كابت النقي لتوليد مستقرة الرفيع المستوى نخاع العظم الخيمرية في الفئران

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

تسليم داخل الأوعية الدموية من البيولوجيات إلى الكلى الجرذ

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

تقييم لوظيفة الكلي في نماذج الماوس من المرض الكبيبي

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

الكشف على أساس الصفائح الدموية من أكسيد النيتريك في الدم عن طريق قياس الفسفرة بسب

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...