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 vaporizer...

<ol>
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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>Arai-Gaun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+oxygenase-1+induced+in+muller+cells+plays+a+protective+role+in+retinal+ischemia-reperfusion+injury+in+rats.">Heme oxygenase-1 induced in muller cells plays a protective role in retinal ischemia-reperfusion injury in rats.</a> Invest Ophthalmol Vis Sci. 45, 4226-4232 (2004).</li><li>Bainbridge, J. W., Tan, M. H., Ali, R. R. <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+progress+and+prospects:+the+eye.">Gene therapy progress and prospects: the eye.</a> Gene Ther. 13, 1191-1197 (2006).</li><li>Polo, A. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+delivery+of+brain-derived+neurotrophic+factor+by+adenovirus-infected+Muller+cells+temporarily+rescues+injured+retinal+ganglion+cells.">Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected Muller cells temporarily rescues injured retinal ganglion cells.</a> Proc Natl Acad Sci U S A. 95, 3978-3983 (1998).</li><li>Herard, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+targeted+against+amyloid+precursor+protein+impairs+synaptic+activity+in+vivo.">siRNA targeted against amyloid precursor protein impairs synaptic activity in vivo.</a> Neurobiol Aging. 27, 1740-1750 (2006).</li><li>Koeberle, P. 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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>Naik, R., Mukhopadhyay, A., Ganguli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+the+retina:+focus+on+non-viral+approaches.">Gene delivery to the retina: focus on non-viral approaches.</a> Drug Discov Today. 14, 306-315 (2009).</li><li>van Adel, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+ciliary+neurotrophic+factor+via+lentiviral-mediated+transfer+protects+axotomized+retinal+ganglion+cells+for+an+extended+period+of+time.">Delivery of ciliary neurotrophic factor via lentiviral-mediated transfer protects axotomized retinal ganglion cells for an extended period of time.</a> Hum Gene Ther. 14, 103-115 (2003).</li><li>Alexander, J. J., Hauswirth, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viral+vectors+and+the+retina.">Adeno-associated viral vectors and the retina.</a> Adv Exp Med Biol. 613, 121-128 (2008).</li><li>Allocca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+gene+transfer+for+retinal+diseases.">AAV-mediated gene transfer for retinal diseases.</a> Expert Opin Biol Ther. 6, 1279-1294 (2006).</li><li>Surace, E. M., Auricchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatility+of+AAV+vectors+for+retinal+gene+transfer.">Versatility of AAV vectors for retinal gene transfer.</a> Vision Res. 48, 353-359 (2008).</li><li>Garcia-Valenzuela, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon-mediated+gene+transfer+of+retinal+ganglion+cells+in+vivo.">Axon-mediated gene transfer of retinal ganglion cells in vivo.</a> J Neurobiol. 32, 111-122 (1997).</li><li>Koeberle, P. D., Wang, Y., Schlichter, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv1.1+and+Kv1.3+channels+contribute+to+the+degeneration+of+retinal+ganglion+cells+after+optic+nerve+transection+in+vivo.">Kv1.1 and Kv1.3 channels contribute to the degeneration of retinal ganglion cells after optic nerve transection in vivo.</a> Cell Death Differ. 17, 134-144 (2010).</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>Leon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+injury+stimulates+axon+regeneration+in+the+mature+rat+optic+nerve.">Lens injury stimulates axon regeneration in the mature rat optic nerve.</a> J Neurosci. 20, 4615-4626 (2000).</li><li>Mansour-Robaey, S. <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+ocular+injury+and+administration+of+brain-derived+neurotrophic+factor+on+survival+and+regrowth+of+axotomized+retinal+ganglion+cells.">Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells.</a> Proc Natl Acad Sci U S A. 91, 1632-1636 (1994).</li><li>D'Onofrio, P. M., Magharious, M. M., Koeberle, P. D. <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+Transection:+A+Model+of+Adult.">Optic Nerve Transection: A Model of Adult.</a> J Vis Exp. , .</li></ol>

Optic nerve transection is a highly reproducible model of adult CNS neuron apoptosis. The experimental manipulations demonstrated in this manuscript permit the study of the mechanisms of RGC apoptosis after injury.
Intraocular injections are useful for global targeting of the retina. This procedure requires some practice, as it is critical not to injure the lens with the tip of the glass pipette. Lens damage has been shown to cause the release of growth factors, altering cell survival and rege...

A correction to the article Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS was made.

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

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<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>
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<td>Alcaine Eye Drops</td>
<td>&nbsp;</td>
<td>Alcon</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>
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<td>Fine tip Dumont forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>10 &mu;l Hamilton Syringe (1701RN; 26s/2&#x201D;/2)</td>
<td>&nbsp;</td>
<td>Hamilton Syringe Co.</td>
<td>80030</td>
<td>&nbsp;</td>
<tr>
<td>1/16 inch Compression Fittings</td>
<td>&nbsp;</td>
<td>Hamilton Syringe Co.</td>
<td>55751-01</td>
<td>&nbsp;</td>
<tr>
<td>1/16 inch OD, 0.010 inch ID, PEEK Tubing</td>
<td>&nbsp;</td>
<td>Supelco, Bellefonte, PA</td>
<td>Z226661</td>
<td>&nbsp;</td>
<tr>
<td>Dual RN Glass Coupler</td>
<td>&nbsp;</td>
<td>Hamilton Syringe Co.</td>
<td>55752-01</td>
<td>&nbsp;</td>
<tr>
<td>Mineral Oil Priming Kit: includes syringe, needles, rubber septa</td>
<td>&nbsp;</td>
<td>Hamilton Syringe Co.</td>
<td>PRMKIT</td>
<td>&nbsp;</td>		</tbody>
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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.

Watch this Scientific Journal Video about Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS at JoVE.com

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

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.

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

Research

JoVE Journal

Neuroscience

13.3K visualizações.  University of Toronto. Transecção do nervo óptico é um modelo amplamente utilizado de lesão do SNC adulto. Este modelo é ideal para a realização de uma série de manipulações experimentais que visam a retina globalmente ou diretamente a população-alvo feridos neuronal de células ganglionares da retina.