PDK is supported by a CIHR operating grant (MOP 86523)

1. Surgical Technique
<ol>
 <li>Experiments should be carried out using aseptic technique and following the animal use protocols of your specific institution. Instruments and materials (solutions, test substances, tracers, needles, etc.) coming into contact with living tissue must be sterile to prevent infection and adverse impacts on animal welfare and potential negative impacts on the study.</li>
</ol>
2. Anesthesia
<ol>
 <li>Rats will be anaesthetized using a veterinary isoflurane...

<ol>
	<li>Bahr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+or+let+die+-+retinal+ganglion+cell+death+and+survival+during+development+and+in+the+lesioned+adult+CNS.">Live or let die - retinal ganglion cell death and survival during development and in the lesioned adult CNS.</a> Trends Neurosci. 23, 483-4890 (2000).</li><li>Isenmann, S., Kretz, A., Cellerino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+determinants+of+retinal+ganglion+cell+development,+survival,+and+regeneration.">Molecular determinants of retinal ganglion cell development, survival, and regeneration.</a> Prog Retin Eye Res. 22, 483-543 (2003).</li><li>Koeberle, P. D., Bahr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+guidance+cues+for+regenerating+axons:+where+have+they+gone.">Growth and guidance cues for regenerating axons: where have they gone.</a> J Neurobiol. 59, 162-180 (2004).</li><li>Weishaupt, J. H., Bahr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+axotomized+retinal+ganglion+cells+as+a+model+for+neuronal+apoptosis+in+the+central+nervous+system+-+molecular+death+and+survival+pathways.">Degeneration of axotomized retinal ganglion cells as a model for neuronal apoptosis in the central nervous system - molecular death and survival pathways.</a> Restor. Neurol. Neurosci. 19, 1-2 (2001).</li><li>Valenzuela, G. a. r. c. i. a., E, S. C. S. h. a. r. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+retinal+ganglion+cells+from+axotomy-induced+apoptosis+through+TRK+oncogene+transfer.">Rescue of retinal ganglion cells from axotomy-induced apoptosis through TRK oncogene transfer.</a> Neuroreport. 9, 3165-3170 (1998).</li><li>Kugler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+axotomized+retinal+ganglion+cells+by+adenoviral+vector+administration+at+the+optic+nerve+stump:+an+in+vivo+model+system+for+the+inhibition+of+neuronal+apoptotic+cell+death.">Transduction of axotomized retinal ganglion cells by adenoviral vector administration at the optic nerve stump: an in vivo model system for the inhibition of neuronal apoptotic cell death.</a> Gene Ther. 6, 1759-1767 (1999).</li><li>Lingor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+apoptosis+mediators+by+RNAi+inhibits+axotomy-induced+retinal+ganglion+cell+death+in+vivo.">Down-regulation of apoptosis mediators by RNAi inhibits axotomy-induced retinal ganglion cell death in vivo.</a> Brain. 128, 550-558 (2005).</li><li>Koeberle, P. D., Gauldie, J., Ball, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+adenoviral-mediated+gene+transfer+of+interleukin-10,+interleukin-4,+and+transforming+growth+factor-beta+on+the+survival+of+axotomized+retinal+ganglion+cells.">Effects of adenoviral-mediated gene transfer of interleukin-10, interleukin-4, and transforming growth factor-beta on the survival of axotomized retinal ganglion cells.</a> Neuroscience. 125, 903-920 (2004).</li><li>Berkelaar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axotomy+results+in+delayed+death+and+apoptosis+of+retinal+ganglion+cells+in+adult+rats.">Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats.</a> J Neurosci. 14, 4368-4374 (1994).</li><li>Villegas-Perez, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influences+of+peripheral+nerve+grafts+on+the+survival+and+regrowth+of+axotomized+retinal+ganglion+cells+in+adult+rats.">Influences of peripheral nerve grafts on the survival and regrowth of axotomized retinal ganglion cells in adult rats.</a> J Neurosci. 8, 265-280 (1988).</li><li>Villegas-Perez, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+protracted+phases+of+retinal+ganglion+cell+loss+follow+axotomy+in+the+optic+nerve+of+adult+rats.">Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats.</a> J Neurobiol. 24, 23-36 (1993).</li><li>Quigley, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+death+in+experimental+glaucoma+and+after+axotomy+occurs+by+apoptosis.">Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis.</a> Invest Ophthalmol Vis Sci. 36, 774-786 (1995).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+transcellular+carbocyanine-labelling+of+rat+retinal+microglia+during+injury-induced+neuronal+degeneration.">Specific transcellular carbocyanine-labelling of rat retinal microglia during injury-induced neuronal degeneration.</a> Neurosci Lett. 127, 108-1012 (1991).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+transcellular+staining+of+microglia+in+the+adult+rat+after+traumatic+degeneration+of+carbocyanine-filled+retinal+ganglion+cells.">Specific transcellular staining of microglia in the adult rat after traumatic degeneration of carbocyanine-filled retinal ganglion cells.</a> Exp Eye Res. 55, 101-117 (1992).</li></ol>

There are many variations of this surgical procedure and several of the steps in this protocol are not necessary. It is only necessary to retract the muscles that overlie the optic nerve in order to gain access to the nerve. However, this results in a very limited working space around the nerve making the critical final stages of transection more difficult. In certain situations it is desirable to transfect the cells from the optic nerve stump and the increased access space afforded by retracting all of the extraocular m...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stereotaxic Frame</td>
<td>&nbsp;</td>
<td>Stoelting, Kopf, WPI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rat Gas Mask</td>
<td>&nbsp;</td>
<td>Stoelting, Kopf, WPI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anesthesia System</td>
<td>&nbsp;</td>
<td>VetEquip</td>
<td>901806</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane (PrAErrane)</td>
<td>&nbsp;</td>
<td>Baxter Corp</td>
<td>DIN 02225875</td>
<td>&nbsp;</td>
<tr>
<td>Surgical Microscope</td>
<td>&nbsp;</td>
<td>WPI, Zeiss, Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorogold -(Hydroxystilbamidine bis(methanesulfonate)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>39286</td>
<td>&nbsp;</td>
<tr>
<td>Gelfoam</td>
<td>&nbsp;</td>
<td>Pharmacia &amp; Upjohn</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tears Naturale P.M.</td>
<td>&nbsp;</td>
<td>Alcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Proviodine</td>
<td>&nbsp;</td>
<td>Medline</td>
<td>MDS093945H</td>
<td>&nbsp;</td>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Fine tip Dumont forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Micro surgical hook</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10062-12</td>
<td>&nbsp;</td>
<tr>
<td>Eye dressing serrated forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11152-10</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #7b sharp curved serrated forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11270-20</td>
<td>&nbsp;</td>
<tr>
<td>Cauterizer</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18010-00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System at JoVE.com

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

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.

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 ...

optic-nerve-transection-model-adult-neuron-apoptosis-central-nervous

Research

JoVE Journal

Neuroscience

20.4K visualizações.  University of Toronto. Transecção do nervo óptico é um modelo amplamente utilizado de lesão do SNC adulto. Noventa por cento das células ganglionares da retina (RGCs) cujos axônios são completamente seccionado (axotomia) morrem dentro de 14 dias após a axotomia. Esse modelo se adequa facilmente a manipulações experimentais e altamente reprodutível.