Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

انظر الجدول رقم 1 لجميع المواد المستخدمة في هذا البروتوكول. تمت الموافقة على التجارب من قبل لجنة رعاية واستخدام الحيوان المؤسسي في جامعة تمبل. 1. الحيوانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحصول wildtype اليرقات البحر الأنقليس (Petromyzon مارينوس L.)، 10 - 14 سم في الطول (4-7 سنة) من مصادر تغذية بحيرة ميشيغان، من روافد لنهر ديلاوير (بنسلفانيا) أو تيارات في ولاية ماين. </li><li style=";text-align:right;direction:rtl"> في المختبر، والحفاظ على اليرقات في مجموعات من 50-100 الحيوانات في خزانات المياه العذبة 50 جالون عند 16 درجة مئوية. خط الدبابات مع بوصة من الحصى لليرقات لتحفر في وتصفية المياه الفحم واشترط بالحجارة الهواء لمدة 48 ساعة على الأقل لإزالة الكلور وغيرها من الشوائب قبل وضع الأنقليس فيها. ليست هناك حاجة لتغذية الحيوانات، والتي تدوير المخلفات الخاصة بهم، حتى يومنا الاستخدام. </li></ol> 2. الكامل العمود الفقريTransection الحبل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل جراحة الحبل الشوكي إعداد حل الجلكى قارع الأجراس مع التشكيل التالي: 110 مم كلوريد الصوديوم، 2.1 ملي بوكل، 2.6 مم CaCl 2، 1.8 ملم MgCl 2 و 10 ملي تريس العازلة؛ ودرجة الحموضة 7.4. </li><li style=";text-align:right;direction:rtl"> لأداء transection الحبل الشوكي، ووضع اليرقات في وعاء صغير ليتم تخدير مع 1٪ تريكين methanesulfonate الجليد في حل قارع الأجراس البارد. </li><li style=";text-align:right;direction:rtl"> مرة واحدة مكان تخدير الحيوانات في طبق بتري تملأ نصف مع Sylgard (سيليكون) وتستخدم 4 دبابيس الحشرات (0.15 مم) لعقد الحيوانات في موقف لعملية جراحية (يتم وضع واحد في خطم الحيوان، واحدة في ذيل واثنين على مستوى الخيشومية الموافق 5). ملء طبق بتري تقريبا إلى أعلى مع حل قارع الأجراس. وضع الطبق مع اليرقة على الجليد أثناء أداء الجراحة تحت stereomicroscope. </li><li style=";text-align:right;direction:rtl"> لأداء transection الحبل الشوكي على ظهري، خط الوسط، وجعل شق طولي مع مشرط (# 11) في مستوى ال 5الخيشومية لتصور الحبل الشوكي. في البداية الجلد مفتوح عن طريق إدراج غيض من شفرة مشرط مواجهة ومن ثم يتم قطع الأنسجة العضلية بلطف شديد حتى يمكن رؤية الحبل الشوكي من فوق. أزياء اثنين من السنانير دبابيس الحشرات واستخدامها لعقد الجدران مفتوحة أثناء تنفيذ transection الحبل الشوكي. </li><li style=";text-align:right;direction:rtl"> تماما القطع في الحبل الشوكي في مستوى الخيشومية الموافق 5 باستخدام زوج من مقص (Castroviejo 8 #)، إدراج مقص عموديا إلى الحبل الشوكي. الحبل الشوكي لابد مقطوع تماما مع قطع نظيفة احد. تصور الحبل الشوكي قطع نهايات تحت stereomicroscope للتأكد من أن الحبل الشوكي تم مقطوع تماما. ثم، إعادة ترتيب قطع طرفي الحبل الشوكي بعد transection باستخدام زوج من ملقط (رقم 5). </li><li style=";text-align:right;direction:rtl"> بعد أداء transection الحبل الشوكي إغلاق الجرح في جدار الجسم الظهرية مع زوج من ملقط غرامة ووضع اليرقات على الجليد لمدة 1 ساعة للسماح للر الجرحس تجف. وضع قطعة من ورق الترشيح بين الجليد واليرقات ونقع عليه مع حل الجلكى قارع الأجراس. درجة حرارة منخفضة تحافظ على الحيوانات الحية في الوقت نفسه أنه يتيح الجرح للهواء الجاف، وبالتالي يساعد على منع الالتهابات. </li><li style=";text-align:right;direction:rtl"> ثم نقل اليرقات إلى خزانات صغيرة (1 يرقة لكل خزان) مع الماء البارد الجليد لمدة 24 ساعة. </li><li style=";text-align:right;direction:rtl"> دراسة lesioned يرقات 1 يوم بعد transection الشوكي لتأكيد سلوكيا أنه لا يوجد الذيلية الحركة إلى موقع الإصابة. ويعتبر الحبل الشوكي transection كاملة إذا اليرقة يمكن أن تتحرك إلا منقاري رأسه إلى موقع الإصابة (على مستوى الخيشومية الموافق 5). إذا كان هذا هو الحال اليرقات ويمكن الآن أن يتم تحويلها إلى خزانات (1 يرقة لكل خزان) مع المياه العذبة في درجة حرارة الغرفة (23 ° C) للسماح لهم لاستعادة لحظات زمنية بالتوقيت الذي تم اختياره. </li><li style=";text-align:right;direction:rtl"> خلال فترة النقاهة تغيير الماء كل 2 إلى 3 أيام. </li></ol> 3. تشريح الدماغ وإعدادالدماغ لالتهجين في الموقع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في الوقت المطلوب نقاط بعد الإصابة إعادة تخدير اليرقات البحر الأنقليس ومكان وارجاعهم في طبق بتري مع حل قارع الأجراس لإجراء تشريح الدماغ على النحو الوارد أعلاه. </li><li style=";text-align:right;direction:rtl"> لتشريح الدماغ خارج، تأكد أولا شق عرضية مع مشرط بين فتحة الأنف والجهاز الصنوبرية. من هنا تبدأ لقطع الأنسجة المحيطة بالمخ باستخدام زوج من مقص Castroviejo حين عقد مع زوج من ملقط. </li><li style=";text-align:right;direction:rtl"> مرة واحدة يتعرض لها المخ، وإزالة الضفيرة المشيمية باستخدام زوج من ملقط واستخدام السنانير لنشر رأس الحيوان وفضح أعصاب في الجمجمة. ثم، وقطع الحبل الشوكي عرضيا مع مقص Castroviejo. عقد الحبل الشوكي مع زوج من ملقط مع استخدام زوج آخر من ملقط لقطع الأعصاب القحفية لتشريح الدماغ بها. </li><li style=";text-align:right;direction:rtl"> بعد تشريح، ووضع الدماغ على قطعة صغيرة من السيليكون والاحتفاظ بها في موقف بإدخال 2 الإضافيةإلخ الدبابيس في بصيلات الشم و 1 في الحبل الشوكي. ثم، وقطع الخلفي والملتقيات cerebrotectal من الدماغ على طول خط الوسط الظهرية مع مقص وصرف لوحات الجناحية أفقيا وارجاعهم شقة للسيليكون مع 4 دبابيس الحشرات (الشكل 2). </li><li style=";text-align:right;direction:rtl"> نقل قطعة من السيليكون مع الدماغ إلى أنبوب يحتوي بارافورمالدهيد 4٪ في الفوسفات مخزنة المالحة (الرقم الهيدروجيني 7.4) لتكون ثابتة في غرفة تيمباراتوري لمدة 3 ساعة على محور دوار. </li><li style=";text-align:right;direction:rtl"> بعد التثبيت استخدام الدماغ لwholemount في الموقع التهجين كما هو موضح سابقا (سوين وآخرون، 1994). يجب تشغيل العقول من السيطرة أو صورية تعمل الحيوانات دائما بالتوازي مع العقول من الحيوانات المصابة. الدماغ اليرقات wholemounted يمكن دراستها عن طريق التهجين في الموقع بسبب شكله المسطح ورقيقة وأيضا بسبب غياب 10 المايلين، مما يجعل الدماغ شفافة إلى حد ما. </li></ol>

<ol>
	<li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury.">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li><li>Davis, G. R., McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey.">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li><li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons.">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li><li>Shifman, M. I., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection.">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li><li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+lamprey+UNC5+receptors+cDNA+cloning,+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development.">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li><li>Shifman, M. I., Yumu, l. R. E., Laramore, C., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey.">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li><li>Busch, D. J., Morgan, 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=Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury.">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li><li>Shifman, M. I., Zhang, G., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys.">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li><li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, M. 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+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults.">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li><li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+myelin+sheaths:+both+lamprey+and+hagfish+lack+myelin.">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li><li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys.">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li></ol>

The authors declare that they have no competing financial interests.

Here we present a detailed protocol to perform a complete spinal cord transection and posterior brain dissection in larval sea lampreys. This procedure allows analyzing differences in gene expression between identifiable spinal cord projecting neurons after spinal cord injury by means of a whole-mount brain in situ hybridization. The critical step in the procedure is the correct performance of a complete spinal cord transection, which can be controlled by observing the cut ends of the spinal cord under the stereomicrocop...

في الثدييات إصابة الحبل الشوكي (النخاع الشوكي) هو شرط المدمرة التي تؤدي إلى فقدان دائم للوظيفة تحت موقع الإصابة لمحاور جرح لا تتجدد من خلال منطقة الصدمة وإعادة الاتصال إلى أهدافهم المناسبة. على عكس الثدييات، الأنقليس التعافي تنقل بعد إصابة الحبل الشوكي كاملة. 1 ومن المثير للاهتمام، الأنقليس لديها مجموعة من 36 الحبل الشوكي إسقاط الخلايا العصبية التي يمكن تحديدها بشكل فردي في جبل لالجامع الاستعدادات الدماغ بسبب حجمها الكبير 2،3 (الشكل 1) . وaxotomized كل من هذه الخلايا العصبية في العمود الفقري عن طريق إسقاط كامل transection الحبل الشوكي على مستوى عال. وقد أظهرت الدراسات السابقة من مجموعتنا وآخرون أنه حتى في وجود الانتعاش وظيفية بعد اصابات النخاع الشوكي بعض هذه الخلايا العصبية تظهر قدرة التجدد منخفضة جدا (أنها تعتبر &quot;regenerators سيئة&quot;)، في حين أن آخرين تتجدد عادة محور عصبي من خلال موقع الإصابة (أنها تعتبر &quot;زregenerators العود &quot;). 2،3 هذه الخاصية تجعل الأنقليس نموذجا الفقاريات مثيرة للاهتمام لدراسة الاختلافات في التعبير الجيني بين الاسترجاع الجيد والسيئ إسقاط الخلايا العصبية في العمود الفقري والتي بدورها سوف تؤدي إلى اختلافات في القدرة على التجدد من الخلايا العصبية الذاتية التي تحاول تجديد محاور عصبية في نفس البيئة خارجي. 1 باستخدام هذا النموذج أظهرنا سابقا أن الخلايا العصبية مع انخفاض القدرة على التجدد المعرض التعبير عن مستقبلات الجزيء التوجيه محور عصبي مثل UNC5 4،5 وneogenin، 6 الذي توسط في العمل المثبطة من netrin وRGM ​​التوالي إسقاط العمود الفقري. بالإضافة إلى ذلك، باستخدام هذا الأسلوب وقد أظهرت مجموعتنا أيضا أن فقط regenerators جيدة تظهر انتعاشا للتعبير عن neurofilaments بعد إصابة وأثناء عملية التجدد. مؤخرا، أظهرت بوش ومورغان 7 بواسطة المناعي أن تظهر regenerators سيئة على الزيادهإد التعبير عن synuclein بعد الإصابة، التي ارتبط بها الكتاب إلى حقيقة أن &quot;المجداد سيئة&quot; إسقاط الخلايا العصبية في العمود الفقري يموتون ببطء بعد استكمال الحبل الشوكي transection 5،7،8. لذلك، برز نموذج الجلكى اصابة في الحبل الشوكي كاملة كنموذج مفيد جدا لفهم ما يجعل الحبل الشوكي والخلايا العصبية إسقاط &quot;المجداد سيئة&quot; بعد axotomy. لإجراء دراسات لدينا ونحن أداء كاملة الشوكي transection الحبل بروتوكول جراحة وتشريح الدماغ الخلفي عند نقاط الوقت المطلوب بعد إصابة لأداء wholemount التهجين في الموقع. في المقالة المنهجية الحالية نقدم بروتوكول مفصلة عن الأداء السليم لعملية جراحية في العمود الفقري إصابة الحبل كاملة في الأنقليس اليرقات، وصيانة لاحقة من الحيوانات وتشريح الدماغ النهائي وإعداد الدماغ لwholemount التهجين في الموقع. بروتوكول مفصل لPErform على wholemount في الموقع التهجين في الدماغ من الأنقليس اليرقات وقد ذكر سابقا. 9 وبالإضافة إلى ذلك، فإن هذا البروتوكول لإصابة الحبل الشوكي وتشريح الدماغ يمكن أن تستخدم أيضا ثم لمعالجة أدمغة لالمناعية النسيجية أو أساليب أخرى.

<table border="0" cellpadding="0" cellspacing="0" width="280">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Name of the reagent</td>
			<td style="width:83px;">Company</td>
			<td style="width:64px;">Catalogue number</td>
			<td style="width:71px;">Comments (optional)</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:64px;">Tricaine methane sulfonate</td>
			<td style="width:83px;">Spectrum</td>
			<td style="width:64px;">TR108</td>
			<td style="width:71px;">Benzocaine saturated solution in PBS for sacrifice</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Scalpel #3</td>
			<td style="width:83px;">Fine Science Tools (FST)</td>
			<td style="width:64px;">10003-12</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Blades for scalpel: #11</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">10011-00</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Castroviejo scissors #8</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">15002-08</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:64px;">Forceps #4 &#38; #5</td>
			<td style="width:83px;">Dumont, Switzerland</td>
			<td style="width:64px;">Roboz RS4955</td>
			<td style="width:71px;">#4 for dissection of Spinal cord; #5 for stripping menninges</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Dissecting Microscope</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:64px;">SZ51</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:64px;">Sylgard</td>
			<td rowspan="2" style="width:83px;">Dow Corning Co.</td>
			<td rowspan="2" style="width:64px;">184</td>
			<td rowspan="2" style="width:71px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="168">
			<td height="189" rowspan="2" style="height:189px;width:64px;">Insect pins 0.15, 0.20 mm</td>
			<td rowspan="2" style="width:83px;">Austerlitz</td>
			<td rowspan="2" style="width:64px;">No catalogue #</td>
			<td rowspan="2" style="width:71px;">0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">7 ml HDPE Scintillation Tubes with Caps</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td style="width:64px;">03-337-1</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Paraformaldehyde 16%</td>
			<td style="width:83px;">Electron Microscopy Science (EMS)</td>
			<td style="width:64px;">19210</td>
			<td style="width:71px;">Dilute to 4% in PBS</td>
		</tr>
	</tbody>
</table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

بعد اصابة كاملة في النخاع الشوكي، والشلل الأنقليس البحر في البداية دون مستوى transection. ومع ذلك، فإنها استعادة تنقل بعد عدة أسابيع، وهذا يرافقه تجديد مسافة قصيرة (بضعة ملم) نخاعي مخصوص من المحاور والمحاور إسقاط العمود الفقري، من جذع الدماغ. بين 36 الخلايا العصبية في العمود الفقري إسقاط-تعريفية كبيرة، وبعضها regenerators جيدة والبعض الآخر سيئة regenerators. يمكن بسهولة تحديد هذه الخلايا العصبية في الجهاز العصبي المركزي الاستعدادات wholemount. من أجل فهم الآليات العصبية الجوهرية التي تحول دون محاباة أو محوار تجديد بعد إصابة في الجهاز العصبي المركزي الفقاريات، نحن تحديد الاختلافات في التعبير الجيني بين regenerators الجيدة والسيئة، وكيف يتأثر التعبير transection الحبل الشوكي. وتوضح هذه الورقة تقنيات اليرقات الإسكان وتحولت مؤخرا الأنقليس البحر الكبار في خزانات المياه العذبة، وإنتاج كاملة transections الحبل الشوكي تحت الرؤية المجهرية، وإعداد رعين وwholemounts الحبل الشوكي لالتهجين في الموقع. باختصار، يتم الاحتفاظ الحيوانات في 16 &#160; ° C وتخدير في 1٪ بنزوكاين في الجلكى قارع الأجراس. ومقطوع الحبل الشوكي مع مقص قطع القزحية عبر نهج الظهرية ويسمح للحيوان لاسترداد في خزانات المياه العذبة في 23 درجة مئوية. لالتهجين في الموقع، وreanesthetized الحيوانات والدماغ والحبل إزالة عبر نهج الظهرية.

As an example of the results that can be obtained when using this method, representative images of wholemounted brains showing the expression of the neogenin transcripts in identifiable spinal cord-projecting neurons of control and 2 weeks post lesion larval sea lampreys are shown in Figure 2. The readers are referred to a previous study6 reporting the relationship between the expression of neogenin after a complete spinal cord transection and the regenerative ability of the identifiable spina...

Watch this Scientific Journal Video about Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey at JoVE.com

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

إصابة الحبل الشوكي الكامل وبروتوكول تشريح الدماغ لاحقا Wholemount<em&gt; في الموقع</em&gt; التهجين في اليرقات البحر الجلكى

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

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Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

9.8K Views.  University of Edinburgh. Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Video: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

تشريح والثقافة من الخلايا العصبية من النخاع الشوكي صواري الجنينية

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

نقرا الإسفار في الموقع التهجين في أقسام المخ طازجة

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

الوحشي قرع السائل : نموذج لإصابات في الدماغ لدى الفئران

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Transection الحبل الشوكي في اليرقات الزرد

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

ل<em&gt; فيفو السابقين</em&gt; يسببها الليزر اصابات الحبل الشوكي نموذج لتقييم آليات محور عصبي تنكس في الوقت الحقيقي

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

الزرد<em&gt; في الموقع</em&gt; الحبل الشوكي التحضير للتسجيلات الكهربية من العمود الفقري الحسية والحركية العصبية

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

التصوير النشاط العصبي في القشرة الأولية Somatosensory باستخدام Thy1-GCaMP6s الفئران المحورة وراثيا

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

فيفو في الأسلوب الثنائي-اللون لتصوير الأوعية الدموية ديناميات بعد إصابة الحبل الشوكي كونتوسيفي

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

التسليم داخل الشرايين للخلايا الجذعية العصبية إلى الدماغ الفئران والفأر: تطبيق على الإقفاري الدماغي

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

تحريض كامل من إصابة الحبل الشوكي من نوع الترانصيب في الفئران

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...