وأيد هذه الدراسة من قبل معهد بحوث أمراض العيون أستراليا (ORIA). نشكر الأستاذ Algis Vingrys والدكتور بانغ بوي، جامعة ملبورن، لمساعدتنا في البداية لتطوير تقنية تسجيل VEP.

وأجريت جميع الإجراءات التي تنطوي على الحيوانات وفقا للقانون الاسترالي من الممارسة لرعاية واستخدام الحيوانات لأغراض علمية والمبادئ التوجيهية لبيان ARVO لاستخدام الحيوانات في العيون والبحوث الرؤية، وتمت الموافقة من قبل: بيان الأخلاق لجنة أخلاقيات الحيوان من جامعة ماكواري. 1. VEP الكهربائي زرع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الحيوانات مع حقن داخل الصفاق من الكيتامين (75 ملغ / كلغ) وmedetomidine (0.5 ملغ / كلغ). ملاحظة: بعد التخدير، ومراقبة ردود الفعل انسحاب (اختبار قرصة، منعكسة القرنية وجفني، الخ) وغيابها مؤشرا لبدء عملية جراحية. باستمرار مراقبة الحيوانات في جميع أنحاء الجراحة وإدارة المخدرات مخدر إضافية (10٪ من جرعة الكيتامين الأولية في أعلى متابعة) في حالة وجود ردود الفعل. وتستخدم الجرذان الكبار (&gt; 12 أسبوعا) في التجارب. </li><li style=";text-align:right;direction:rtl"> حلق الجلد من منطقة العمليات الجراحية.وضع الحيوان على وسادة ارتفاع درجة حرارة (37 درجة مئوية) للحفاظ على درجة حرارة الجسم أثناء الجراحة. تحضير الجلد من خلال التطبيق الموضعي للبوفيدون اليود. تطبيق اللف الجراحي. تطبيق مرهم موضعي لمنع جفاف القرنية تحت التخدير العام. الحفاظ على عقامة باستخدام أدوات معقمة. </li><li style=";text-align:right;direction:rtl"> جعل شق الجلد الطولي في خط الوسط من جلد الرأس. مسح النسيج الضام لتحقيق التعرض جيدة من الجمجمة. </li><li style=";text-align:right;direction:rtl"> بعناية، حفر ثقوب لدغ صغيرة يدويا باستخدام الحفر اليد الصغيرة في 7 مم وراء bregma و 3 مم الوحشي على خط الوسط. </li><li style=";text-align:right;direction:rtl"> أقطاب المسمار الزرع من خلال الجمجمة في القشرة (منطقة 17)، اختراق القشرة على عمق حوالي 0.5 مم. زرع قطب كهربائي المسمار إشارة على خط الوسط 3 مم منقاري إلى bregma. تطبيق الاسمنت الأسنان ليغلف وإصلاح مسامير (غير مطلوب دائما). </li><li style=";text-align:right;direction:rtl"> خياطة الجلد من الرأس، وإدارة مرهم مضاد حيوي على الجلد والسماح للحيوان لريكوالنسخة من التخدير على لوحة ظاهرة الاحتباس. ملاحظة: بدلا من ذلك، الأقطاب الكهربائية يمكن تركها مكشوفة، لذلك لا يحتاج أن الجلد لإعادة فتح في كل تسجيل. فور وقف عملية جراحية ولكن قبل الاستعادة مخدر، وإدارة الدواء غير الستيرويدية المضادة للالتهابات (NSAID) (إن لم تكن تدار قبل الجراحة) أو مسكن أفيوني. مراقبة الحيوانات باستمرار حتى الشفاء التام من التخدير والعيادات الخارجية بالكامل. </li><li style=";text-align:right;direction:rtl"> تسمح على الأقل 1 أسبوع للحيوانات للتعافي من عملية جراحية قبل التسجيل VEP. </li></ol> 2. حقن العصب البصري <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الحيوان، وإعداد البشرة وتطبيق اللف على النحو الوارد أعلاه (1.1 و 1.2). </li><li style=";text-align:right;direction:rtl"> جعل 1- 1.5 سم شق في الجلد فوق المدار من العين تم اختيارها عشوائيا. فتح الأنسجة تحت الجلد لتصل إلى تجويف المداري باستخدام مقص القزحية الجميلة. فتح الملتحمة والأمامي كبسولة تينون تحت المجهر التشغيل. </li><li style=";text-align:right;direction:rtl"> التراجع عن المقلةالعضلات والغدد الدمعية intraobital لفضح ما يقرب من 3 ملم طول العصب البصري. فتح الجافية والمواد العنكبوتية طبقات حول العصب البصري طوليا باستخدام شفرة العيون. </li><li style=";text-align:right;direction:rtl"> إدراج ماصة الزجاج في العصب البصري على مسافة 2 مم الخلفي إلى العالم. يتم إرفاق micropipette الزجاج لحقنة هاملتون. </li><li style=";text-align:right;direction:rtl"> حقن 1٪ ليزوليسيتين (0،4-1،0 ميكرولتر، مع 0.02٪ الأزرق ايفان والتي لا يكون لها آثار على المايلين) ببطء في العصب تقريبا على مدى فترة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> خياطة شق الجلد. تطبيق مرهم مضاد حيوي لمنع العدوى. ويمكن تقديم عيون زميل والضوابط الداخلية للتسجيلات الكهربية. </li><li style=";text-align:right;direction:rtl"> وضع الحيوانات على لوحة ظاهرة الاحتباس للتعافي من التخدير. </li></ol> 3. VEP تسجيل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الحيوان وإعداد الجلد مثل 1.1 و 1.2. ملاحظة: أقل جرعة من التخدير يمكن استخدامها لريكو الكهربيةrding (الكيتامين 40 ملغ / كغ وmedetomidine 0.25 ملغ / كلغ). </li><li style=";text-align:right;direction:rtl"> وضع فأر في غرفة مظلمة واتركه على التكيف مع الظلام ل5-30 دقيقة. في بعض الحالات الفئران يمكن أن يكون على التوالي الظلام O تكييفها / N للالرؤية الليلية أو ضوء تكييفها للتسجيلات ضيائية VEP 8. </li><li style=";text-align:right;direction:rtl"> الحفاظ على درجة حرارة الجسم عند 37 ± 0.5 ° C من قبل النظام بطانية homoeothermic مع لجنة التحقيق مقياس الحرارة المستقيم. </li><li style=";text-align:right;direction:rtl"> تمدد التلاميذ مع 1.0٪ قطرات تروبيكاميد العين. فتح الجلد فوق الجمجمة للوصول إلى أقطاب المسمار في الموقع الطبيعي وضعت مسبقا. </li><li style=";text-align:right;direction:rtl"> ربط المسمار على القشرة البصرية المقابل للعين حفز والمسمار إشارة إلى مكبر للصوت. إدراج القطب الإبرة في ذيل مثل الأرض. قياس والحفاظ على مقاومة الكهربائي أقل من 5 أوم. </li><li style=";text-align:right;direction:rtl"> وضع مشجعا مصغرة فريق Ganzfeld مباشرة على الجلد حول الجفون لتوفير العين متفوقة العزلة 7. إضاءة للمشجعا تحتاج إلى معايرة مسبقاقبل مضواء. </li><li style=";text-align:right;direction:rtl"> تقديم تحفيز ضوئي من خلال ومضات ضوء 100 مرة بتردد 1 هرتز، مع إعدادات التصفية الفرقة تمرير المنخفضة والعالية من 1 و 100 هرتز، على التوالي. معدل أخذ العينات إشارة هو في 5 كيلو هرتز. ملاحظة: يجب أخذ عينات إشارات على الأقل في حوالي 250-300 هرتز للتأكد من أن أكثر من اثنين من العينات التي تم جمعها خلال كل دورة. </li><li style=";text-align:right;direction:rtl"> خياطة الجلد إلى الوراء والحفاظ على الحيوانات على لوحة ظاهرة الاحتباس للتعافي من التخدير. تسجيل يمكن تسجيل مرارا وتكرارا على الحيوانات الفردية لرصد التغيرات الوظيفية على مدى فترة من الزمن. </li><li style=";text-align:right;direction:rtl"> في نهاية المرحلة، إدارة حقن جرعة زائدة من الصوديوم بنتوباربيتون (100 ملغ / كغ، والملكية الفكرية) إلى الموت ببطء الحيوان. تأكيد القتل الرحيم التي كتبها سكتة قلبية، توقف التنفس وانخفاض في درجة حرارة الجسم. </li></ol> 4. إعداد الأنسجة والأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة الأعصاب البصرية من الحيوانات الموت الرحيم تحت المجهر وإصلاح الأنسجة في 1٪ بارافورمالدهيد O / N. </li> &lt;لى&gt; غسل الأنسجة تماما باستخدام محلول ملحي. علاج الأنسجة في معالج النسيج الآلي وتغرس في البارافين. أقسام قطع (5-10 ميكرون) باستخدام مشراح الدوارة. ملاحظة: للحصول على دراسة المناعية، وإصلاح الأنسجة في 1٪ امتصاص العرق، ويغسل بمحلول ملحي واحتضان مع 30٪ سكروز O / N. الأنسجة تغرس في أكتوبر التضمين المتوسطة وتجعل cryosections باستخدام ناظم البرد. </li><li style=";text-align:right;direction:rtl"> احتضان المقاطع في 0.1٪ محلول أزرق سريع مثل Luxol (في 95٪ من الإيثانول) O / N عند 56 ° C. تمييز المقاطع في 0.05٪ كربونات الليثيوم لمدة 30 ثانية ثم الايثانول 70٪ لمدة 30 ثانية أخرى. أخيرا، مباين في 0.1٪ محلول الكريزيل البنفسجي لمدة 30 ثانية قبل تركيب الأبواب. استخدام تلطيخ الأزرق سريع لتحديد المايلين في العصب البصري 5. </li></ol>

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+reproducibility+of+VEP+recording+in+rats:+electrodes,+stimulus+source+and+peak+analysis.">Improving reproducibility of VEP recording in rats: electrodes, stimulus source and peak analysis.</a> Doc Ophthalmol. 123 (2), 109-119 (2011).</li><li>Heiduschka, P., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+visual+function+in+pigmented+and+albino+rats+by+electroretinography+and+visual+evoked+potentials.">Comparison of visual function in pigmented and albino rats by electroretinography and visual evoked potentials.</a> Graefes Arch Clin Exp Ophthalmol. 246 (11), 1559-1573 (2008).</li><li>You, Y., Thie, J., Klistorner, A., Gupta, V. K., Graham, 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=Normalization+of+visual+evoked+potentials+using+underlying+electroencephalogram+levels+improves+amplitude+reproducibility+in+rats.">Normalization of visual evoked potentials using underlying electroencephalogram levels improves amplitude reproducibility in rats.</a> Invest Ophthalmol Vis Sci. 53 (3), 1473-1478 (2012).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+optic+nerve+diseases.">Animal models of optic nerve diseases.</a> Eye (Lond). 18 (11), 1066-1074 (2004).</li><li>Henry, K. R., Rhoades, R. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature-dependent+and+independent+actions+of+chlordimeform+on+visual+evoked+potentials+and+axonal+transport+in+optic+system+of+rat.">Body temperature-dependent and independent actions of chlordimeform on visual evoked potentials and axonal transport in optic system of rat.</a> Neuropharmacology. 24 (8), 743-749 (1985).</li><li>Hetzler, B. E., Boyes, W. K., Creason, J. P., Dyer, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+changes+in+visual+evoked+potentials+of+rats.">Temperature-dependent changes in visual evoked potentials of rats.</a> Electroencephalogr Clin Neurophysiol. 70 (2), 137-154 (1988).</li><li>Mitchell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+lysolecithin+on+non-myelinated+axons+in+vitro.">The effects of lysolecithin on non-myelinated axons in vitro.</a> Acta Neuropathol. 58 (4), 243-248 (1982).</li><li>Meyer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+neuronal+apoptosis+in+a+rat+model+of+multiple+sclerosis.">Acute neuronal apoptosis in a rat model of multiple sclerosis.</a> J Neurosci. 21 (16), 6214-6220 (2001).</li><li>Lachapelle, F., 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=Failure+of+remyelination+in+the+nonhuman+primate+optic+nerve.">Failure of remyelination in the nonhuman primate optic nerve.</a> Brain Pathol. 15 (3), 198-207 (2005).</li></ol>

None of the authors have competing interests or conflicting interests.

The optic nerve is very susceptible to mechanical damage. Optic nerve crush injury over a duration of 1 s can lead to about 75% loss of RGC over a period of 2 weeks10. Therefore, extreme care is required while performing the surgical procedures. According to the authors&#8217; experience, it is much better to adapt a blunt dissection approach to expose and make way through the tissues around the optic nerve along the orientation of the nerve, rather than penetrating in a perpendicular orientation to the optic ...

Optic neuritis is one of the most common form of optic neuropathy, causing complete or partial loss of vision1. Histologically, it is featured by inflammatory demyelination, retinal ganglion cell axonal loss and varying degrees of remyelination in the optic nerve2. Optic neuritis is usually the manifest onset of multiple sclerosis. The visual evoked potential (VEP) is a non-invasive tool for investigating the function of the visual system. It reflects the post-retinal function from the retina to the primary visual cortex and is affected in many optic nerve disease conditions3. The VEP has been predominantly used in optic neuritis patients to assess the integrity of the visual pathway4. 
The latency of VEP, which reflects the velocity of signal conduction along the visual pathway, is considered to be an accurate measurement of the level of myelin associated changes in the optic nerve5; while the amplitude of VEP is believed to be closely correlated with axonal damage of the retinal ganglion cells (RGC)6. This hypothesis has been fairly well established using the rat model of lysolecithin-induced optic nerve demyelination5. 
Here, we explicate a comprehensive protocol of optic nerve microinjection technique in rodents, which can minimise the surgical manipulation-related damage to the nerve per se as well as to the adjacent tissues such as extraocular muscles and blood vessels. Also, the skull electrode implantation surgery has been described for VEP recording in animals7. The VEP recordings can be repeatedly carried out on animals over a period of time to assess demyelination/remyelination related changes as well as impact on axonal integrity in the optic nerve.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine 100 mg/ml (Ketamil)</td>
			<td>Troy Laboratories</td>
			<td>AC 116</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine 1 mg/ml (Domitor)</td>
			<td>Pfizer</td>
			<td>sc-204073</td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide 1.0% (Mydriacyl)</td>
			<td>Alcon</td>
			<td>sc-202371</td>
			<td></td>
		</tr>
		<tr>
			<td>Homoeothermic blanket system</td>
			<td>Harvard Apparatus</td>
			<td>NC9203819</td>
			<td></td>
		</tr>
		<tr>
			<td>Impedance meter&#160;</td>
			<td>Grass</td>
			<td>F-EZM5</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw electrodes&#160;</td>
			<td>Micro Fasteners</td>
			<td>M1.0&#215;3mm Csk Slot M/T 304 S/S</td>
			<td></td>
		</tr>
		<tr>
			<td>Subdermal needle electrodes&#160;</td>
			<td>Grass</td>
			<td>F-E3M-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid Repair&#160;</td>
			<td>DeguDent GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light-emitting diode&#160;</td>
			<td>Nichia</td>
			<td>NSPG300A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioamplifier</td>
			<td>CWE, Inc.</td>
			<td>BMA-400</td>
			<td></td>
		</tr>
		<tr>
			<td>CED system</td>
			<td>Cambridge Electronic Design, Ltd.</td>
			<td>Power1401</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe&#160;</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysolecithin</td>
			<td>Sigma</td>
			<td>L4129</td>
			<td></td>
		</tr>
		<tr>
			<td>Evan&#8217;s blue&#160;</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

وتظهر آثار استنساخه VEP داخل الدورات في الشكل 1 و تأخير كبير في الكمون N1 يمكن أن ينظر بعد حقن العصب البصري. ويمكن ملاحظة آفات العصب البصري جزئية من إزالة الميالين على أقسام النسيجية باستخدام Luxol تلطيخ الأزرق سريع 5 الشكل 2 يوضح مقطع تمثيلي مع آفة ?...

Watch this Scientific Journal Video about Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination at JoVE.com

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

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 ...

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JoVE Journal

Neuroscience

12.1K Views.  Macquarie University. 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. 

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

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 ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

تحديد أنواع غشاء الميتوكوندريا المحتملة والأوكسجين التفاعلي في الخلايا العصبية الفئران الحية القشرية

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

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.

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 ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

تسجيل وقت واحد من تخطيط كهربية الشبكية والإمكانيات البصرية مستدعى في الفئران تخدير

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

ترانسيكتيون جزئي في العصب البصري في الفئران: نموذج المنشأة مع نهج منطوق جديد لتقييم ثانوي ضمور خلايا الشبكية العقدة

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

تسجيلات المحتملة مقولة البصرية في الفئران باستخدام الجاف غير الغازية متعدد القنوات فروة الرأس الاستشعار EEG

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

نموذج الفئران من إزالة المزيل القشرية الدماغية على نطاق واسع الناجمة عن الحقن داخل الدماغ من السيتوكينات الموالية للالتهابات

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

المحفزات الخاصة بالقشرية البصرية التي تثير الأنماط المورفولوجية المحتملة

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...