This study was supported by NeuralStem, Inc., and Johns Hopkins Project RESTORE.

ملاحظة: تمت الموافقة على جميع الإجراءات الحيوانية من لجنة رعاية الحيوان واستخدام جونز هوبكنز. آلات التخدير تتطلب التفتيش السنوي والمعايرة حسب الضرورة. 1. التخدير وتحديد المواقع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التخدير. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أداء جميع العمليات الجراحية تحت التخدير مع 2-3٪ isofluorane. تأكيد مستوى مناسب من التخدير من قرصة أخمص قدميها، ومعدل التنفس. تأكد من أن الفئران لا تتوانى في استجابة لقرصة أخمص قدميه. ملاحظة: تتوانى يشير التخدير أن يفوت الأوان، وقد يتطلب تخدير أطول قبل بداية أو تركيز isofluorane العالي. معدل التنفس أقل من 1 التنفس كل 2 ثانية بطيئة جدا مما يدل على التخدير مرتفع جدا، وقد تتطلب البرق للتركيز isofluorane. </li><li style=";text-align:right;direction:rtl"> تأمين الحيوان عند الضرورة لتجنب حركة الرأس أثناء العملية. وضع قطرة من يدوكائين على العين الجراحية. استخدام الدموع الاصطناعية كل 10 دقيقة لمنع جفاف بينما تحت التخدير. منححقن البوبرينورفين 0.01 مغ / كغ SC ما قبل الجراحة وبعد ذلك كل 6-8 ساعة حسب الحاجة. </li></ol></li><li style=";text-align:right;direction:rtl"> تحديد المواقع. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع الفئران في إطار التجسيمي والدفء مع وسادة التدفئة. الرطب الفراء فروة الرأس مع الكحول مع الحرص على تجنب التعرض على العينين. استخدام أدوات معقمة وتقنية معقمة للحد من خطر العدوى بعد العمليات الجراحية. </li></ol></li></ol> 2. التحكم في العين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع خياطة 4-0 في الملتحمة الجانبية وربط ذلك مع ما يكفي من خياطة للسماح الجر لطيف. </li></ol> 3. تشريح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تشريح الأولي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل بوصة شق ~ 1 في الجلد المغطي التلال المداري باستخدام حجم 10 مشرط كما هو مبين في الشكل 1. يتراجع عن الجلد واللفافة الكامنة وتشريح بعناية بعيدا اللفافة. لمنع النزيف في العمليات الجراحية، وتجنب قطع الأوعية الدموية في حين تشريح اللفافة. استخدام استراتيجيا ررنصائح القطن ماجد لتوفير الارقاء. </li></ol></li><li style=";text-align:right;direction:rtl"> تشريح أعمق. ملاحظة: مع الجر لطيف على الملتحمة سحب العين السقوط والخروج من مأخذ، وسوف يأتي عضلة المدارية متفوقة في طريقة العرض. من أجل فضح العصب البصري، يجب أن تقطع هذه العضلات وإزالة الدهون الرجعية المدارية. الدهون يمكن التخلص منها ويجب أن لا يتم استبدال بعد الحقن. من هذه النقطة، يجب أن يكون لفافة العصب البصري المرئي وكأنه مجموعة من العصب البصري في حد ذاته، جنبا إلى جنب مع الأوعية الدموية ملفوفة في الجافية (الشكل 1). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إجراء شق صغير في الجافية باستخدام مشرط أو 31 مقياس مشطوف إبرة لجعل ثقب أقل من صدمة. </li></ol></li></ol> 4. ماصة الحاقن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> سحب micropipette الزجاج ليبلغ قطرها 50-100 ميكرون. لتوفير الاستقرار وتركيب micropipette على micromanipulator ونعلق على حقنة هاميلتون متصلة مضخة التسريب. </li><li style=";text-align:right;direction:rtl"> سحب ما يصل حبات (أو الخلايا الجذعية)أعيد في حجم 0،5-1،0 ميكرولتر في micropipette الوراء جنبا إلى جنب مع 0.5 حجم ميكرولتر من حل الميثيل الأزرق قبل وبعد. ملاحظة: ان معدل ضخ المقرر أن 0،5-2 ميكرولتر لكل دقيقة يمنع الصدمات في العصب البصري. </li></ol> 5. حقن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خفض غيض من micropipette على العصب البصري فقط فوق الجافية رصدت. ملاحظة: حركة صغيرة، ولكن انتعش من طرف زجاج في نتائج العصب البصري في أقل الأضرار. كما تبدأ مضخة التسريب، ينبغي للالميثيل صبغة زرقاء تسليط الضوء على منطقة العصب البصري حقن. ينبغي أن تظل الصبغة المحلية داخل العصب البصري دون تسرب في الفضاء تحت العنكبوتية. </li><li style=";text-align:right;direction:rtl"> اتبع الفرقة الثانية للأزرق الميثيل للوقوف عند حقن الخلايا الجذعية اكتمال وتشغيل مضخة التسريب قبالة كما يتم حقن الجرعة الثانية من اللون الأزرق الميثيل. الحفاظ على micropipette لا يزال داخل العصب البصري لمدة 2 دقيقة لكل وحدة تخزين 1 ميكرولتر حقنها لمنع ارتفاعضغط طرد على الانسحاب من micropipette. ملاحظة: هذا أسلوب بديل لتوصيل طرف ماصة لشغل حقنة الهواء التي يمكن التلاعب بها يدويا. وفي كلتا الحالتين، وتجنب استخدام القوة المفرطة للحد من الأضرار التي لحقت الأعصاب البصرية. ونحن نوصي حقن لا يزيد عن 2 ميكرولتر من حجم في كل العصب البصري. </li></ol> 6. متابعة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فوري. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عندما حقن اكتمال، وإزالة micropipette جنبا إلى جنب مع أي نصائح القطن المستخدمة لتوفير الارقاء. خياطة الجلد مع 3-0 الحرير وإزالة خياطة الملتحمة. </li><li style=";text-align:right;direction:rtl"> الحفاظ على الفئران دافئة على وسادة التدفئة حتى خروجها من التخدير. لا تترك حيوان غير المراقب حتى استعاد وعيه أنه كاف للحفاظ على الاستلقاء القصية. لا يعودون الحيوان الذي خضع لعملية جراحية للشركة من الحيوانات الأخرى حتى تعافى تماما. </li><li style=";text-align:right;direction:rtl"> إذا كان الحيوان هو اظهار علامات الألم بما في ذلك الخمول المفرط، وترنح، أو يتنفس جاهدز، إدارة التسكين المناسب مع البوبرينورفين العضلي (0.05 ملغ / كلغ) ما يصل إلى ثلاث مرات يوميا. </li></ol></li><li style=";text-align:right;direction:rtl"> طويل الأمد. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لساعة 24-48 المقبلة، ومراقبة الفئران لمضاعفات الجراحة بما في ذلك تورم أو إفرازات من الجرح أو غيرها من علامات الألم مثل اللفظ، والمظهر منحنية، وعدم النظافة الشخصية، أو لا يأكل. النظر في التشاور مع طبيب بيطري أو القتل الرحيم الأخلاقي عند الضرورة. </li></ol></li></ol>

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	<li>Dahlmann-Noor, A., Vijay, S., Jayaram, H., Limb, A., Khaw, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+and+future+prospects+for+stem+cell+rescue+and+regeneration+of+the+retina+and+optic+nerve.">Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve.</a> Canadian journal of ophthalmology Journal canadien d'ophtalmologie. 45 (4), 333-341 (2010).</li><li>Quigley, H. A., Iglesia, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+to+replace+the+optic+nerve.">Stem cells to replace the optic nerve.</a> Eye. 18 (11), 1085-1088 (2004).</li><li>Ghaffarieh, A., Levin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+disease+and+axon+pathophysiology.">Optic nerve disease and axon pathophysiology.</a> International review of neurobiology. 105, 1-17 (2012).</li><li>Fukui, Y., Hayasaka, S., Bedi, K. S., Ozaki, H. S., Takeuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+the+development+of+the+optic+nerve+in+rats+reared+in+the+dark+during+early+postnatal+life.">Quantitative study of the development of the optic nerve in rats reared in the dark during early postnatal life.</a> Journal of anatomy. 174, 37-47 (1991).</li><li>Celestre, P. C., 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=Minimally+invasive+approaches+to+the+cervical+spine.">Minimally invasive approaches to the cervical spine.</a> The Orthopedic clinics of North America. 43 (1), 137-147 (2012).</li><li>Hallas, B. H., Wells, M. 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+Novel+Technique+for+Multiple+Injections+into+the+Mammalian+Optic+Nerve.">A Novel Technique for Multiple Injections into the Mammalian Optic Nerve.</a> Kopf Carrier. 54, (2001).</li><li>Harvey, A. R., Hellstrom, M., Rodger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+and+transplantation+in+the+retinofugal+pathway.">Gene therapy and transplantation in the retinofugal pathway.</a> Progress in brain research. 175, 151-161 (2009).</li><li>Adachi-Usami, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+neuritis--from+diagnosis+to+optic+nerve+transplantation.">Optic neuritis--from diagnosis to optic nerve transplantation.</a> Nippon Ganka Gakkai zasshi. 104 (12), 841-857 (2000).</li><li>Slater, B. J., Vilson, F. L., Guo, Y., Weinreich, D., Hwang, S., Bernstein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+inflammation+and+demyelination+in+a+rodent+model+of+nonarteritic+anterior+ischemic+optic+neuropathy.">Optic nerve inflammation and demyelination in a rodent model of nonarteritic anterior ischemic optic neuropathy.</a> Investigative ophthalmology &amp; visual science. 54 (13), 7952-7961 (2013).</li><li>Zarbin, M. A., Arlow, T., Ritch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+nanomedicine+for+vision+restoration.">Regenerative nanomedicine for vision restoration.</a> Mayo Clinic proceedings. 88 (12), 1480-1490 (2013).</li></ol>

The authors have no meaningful disclosures to report.

Direct injection into the optic nerve of stem cells or other products intended to facilitate regeneration provides a convenient model compared to other means of injections into the CNS. This technique takes less time, requires less total anesthesia, avoids drilling or removing skull or bone tissue, reduces complications rates and allows for more rapid recovery following surgery. 
The most critical steps in this protocol include: 1. Adequate hemostasis in the surgical field to allow clear visua...

يوفر العصب البصري موقعا مثاليا للنظام العصبي المركزي (CNS) البحوث التجدد بما في ذلك شروط العيون مثل التهاب العصب البصري، والزرق والصدمات النفسية. حقن مجموعة متنوعة من الخلايا الجذعية أثبتت إما فعالية أو أظهرت الوعد في استبدال المايلين المفقود، وزيادة عدد المحاور و / أو الوقاية من الأمراض التنكسية. 1،2 العصب البصري البشري يحتوي على ما يقرب من 1.2 مليون محاور متوازية متجهة من الشبكية إلى تصالبة التي يبلغ قطرها حوالي 3.0-3.5 مم. 3 لنموذج الأمراض التي تصيب الإنسان في المختبر، وقد تم استخدام الفئران في كثير من الأحيان. والعصب البصري الفئران الكبار يحتوي على حوالي 100،000 المحاور داخل يبلغ قطرها حوالي 0.5 مم. 4 واحدة من القيود الرئيسية في الجهاز العصبي المركزي البحوث التجديدي هو الوصول العظم المباشر. المضاعفات والمخاطر الجراحية للحيوان أعلى عندما تتم إزالة الجمجمة أو الفقرات. وعلى غرار فوائدالنهج الحد الأدنى الغازية في العمود الفقري، 5 المباشر حقن العصب البصري دون فتح الجمجمة عرض تخفيض المضاعفات وانتعاش أكثر سرعة. وقد استخدمت هذه التقنية في الدراسات السابقة. 6 وفي هذه المخطوطة والفيديو المصاحبة لها، علينا أن نظهر إجراء المناظير لحقن الخلايا الجذعية في العصب البصري الفئران.

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
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			<td style="width:335px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">Lewis rat</td>
			<td style="width:151px;">Charles River</td>
			<td align="right" style="width:151px;">4</td>
			<td>Any rat strain will work.</td>
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			<td height="21" style="height:21px;width:216px;">Anesthesia machine</td>
			<td style="width:151px;">Surgivet</td>
			<td style="width:151px;">CDS9000</td>
			<td>CDS 9000 Small Animal Anesthesia Machine - Pole Mount</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Infusion pump</td>
			<td style="width:151px;">Stoelting</td>
			<td align="right" style="width:151px;">53129</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:151px;">National Optical</td>
			<td style="width:151px;">409-411-1105</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiber-optic light source</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:151px;">12-562-21</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissection and Stereotaxic Instrument</td>
			<td style="width:151px;">Stoelting</td>
			<td align="right" style="width:151px;">51400</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette Puller</td>
			<td style="width:151px;">Kopf</td>
			<td align="right" style="width:151px;">750</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pipettes</td>
			<td style="width:151px;">World Precision Instruments</td>
			<td style="width:151px;">18150-6</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable scalpel blades</td>
			<td style="width:151px;">Harvard Apparatus</td>
			<td style="width:151px;">810-15-021</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Iridectomy scissors</td>
			<td style="width:151px;">Electron Microscopy Sciences</td>
			<td style="width:151px;">Uniband LA-4XF</td>
			<td></td>
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minimally-invasive technique for injection into rat optic nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

في ختام التجربة، تم التضحية الفئران ومع perfused 4٪ لامتصاص العرق. تم الأعصاب البصرية تشريح بعناية والتي شنت لناظم البرد باجتزاء الشكل 2 يوضح مثال على الفئران العصب البصري كله في الطاقة المنخفضة التي تم حقن صبغة زرقاء ايفانز من أجل رؤية الموقع. السهم يحدد الموقع ...

Watch this Scientific Journal Video about Minimally-invasive Technique for Injection into Rat Optic Nerve at JoVE.com

Minimally-invasive Technique for Injection into Rat Optic Nerve

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

تقنيات الحد الأدنى الغازية لحقن الفئران في العصب البصري

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

minimally-invasive-technique-for-injection-into-rat-optic-nerve

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9.4K Views.  Johns Hopkins University. Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid. 

Video: Minimally-invasive Technique for Injection into Rat Optic Nerve

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

وحدة واحدة في الجسم الحي تسجيلات من chiasm البصرية من الجرذ

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

جبلة المحوار من عزل العصب الوركي الجرذ

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

العصب البصري Transection : نموذج من موت الخلايا المبرمج الخلية العصبية الكبار في الجهاز العصبي المركزي

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

أساليب التلاعب التجريبية بعد Transection العصب البصري الثدييات في الجهاز العصبي المركزي

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

وسحق العصب البصري النموذجي إصابة الفئران لدراسة الخلايا الشبكية بقاء العقدة

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

في الجسم الحي التصوير من الألياف البصرية النزاهة العصب عن طريق التصوير بالرنين المغناطيسي تعزيز التباين في الفئران

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

الحقن في العضل وإلى جانب لوحات السيارات نهاية: نهج المناظير لمكوك الراسمات مباشرة إلى الخلايا العصبية للسيارات

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

البصري مستدعى تسجيل المحتملين في نموذج الفئران التجريبية العصب البصري إزالة الميالين

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Endoscopic Endonasal Trans-sphenoidal Approach: Minimally Invasive Surgery for Pituitary Adenomas

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary lesions. Refinements in surgical techniques, technological advancements, and incorporation of neuronavigation have rendered this surgery minimally invasive. The complication rates of this surgery are very low while excellent results are consistently obtained through this approach.
This paper focuses on the step-by-step surgical approach to pituitary adenomas, which is based on personal experience, and details the results obtained with this minimally invasive surgery.

The aim of this paper is to describe the different steps of the endoscopic endonasal approach to the sella turcica.

النهج بالمنظار اندوناسال ترانس-سفينويدال: الجراحات لاورام الغدد النخامية

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary ...

Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based treatment options for this devastating disease process. In the past decade, a number of minimally invasive surgeries have emerged to address this issue, one of which is endoscopic evacuation. Stereotactic ICH Underwater Blood Aspiration (SCUBA) is a novel endoscopic evacuation technique performed in a fluid-filled cavity using an aspiration system to provide an additional degree of freedom during the procedure. The SCUBA procedure utilizes a suction device, endoscope, and sheath and is divided into two phases. The first phase involves maximal aspiration and minimal irrigation to decrease clot burden. The second phase involves increasing irrigation for visibility, decreasing aspiration strength for targeted aspiration without disturbing the cavity wall, and cauterizing any bleeding vessels. Using the endoscope and aspiration wand, this technique aims to maximize hematoma evacuation while minimizing collateral damage to the surrounding brain. Advantages of the SCUBA technique include the use of a low-profile endoscopic sheath minimizing brain disruption and improved visualization with a fluid-filled cavity rather than an air-filled one.

This paper details the surgical protocol for minimally invasive endoscopic intracerebral hemorrhage evacuation using the SCUBA technique.

الإخلاء النزفي الداخلي المنظاري الحد الأدنى

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based ...