The authors thank Joris Van Asselberghs and Ann Van Santvoort for their excellent technical assistance. Research was funded by the IWT-Vlaanderen (IWT SBO/80020), the FWO Vlaanderen (G.0768.10), by the EC-FP6 program &#39;DiMI&#39; (LSHB-CT-2005-512146), the FP7 RTD project MEFOPA (HEALTH-2009-241791), the FP7 program &#39;INMiND&#39; (HEALTH-F2-2011-278850), the KU Leuven (IOF-KP/07/001, OT/08/052A, IMIR PF/10/017), and the MJFox Foundation (Target validation 2010). A. Van der Perren and C. Casteels are&#160;a postdoctoral fellows of the Flemish Fund of Scientific Research. K. Van Laere is a senior clinical fellow of the Flemish Fund of Scientific Research.

All animal experiments are carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Bioethical Committee of the University of Leuven (Belgium).
1. Recombinant AAV Production and Purification
Note: rAAV vector production and purification was performed by the Leuven Viral Vector Core (LVVC) as previously described17.
<ol>
	<li>Briefly, transfect subconfluent low (&#60;50) passage adherent HEK 293T cells using a 25kD linear poly...

<ol>
	<li>Magen, I., Chesselet, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+of+Parkinson's+disease+The+state+of+the+art.">Genetic mouse models of Parkinson's disease The state of the art.</a> Prog Brain Res. 183, 53-87 (2010).</li><li>Masliah, E., 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=Dopaminergic+loss+and+inclusion+body+formation+in+alpha-synuclein+mice:+implications+for+neurodegenerative+disorders.">Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders.</a> Science. 287, 1265-1269 (2000).</li><li>Freichel, 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=Age-dependent+cognitive+decline+and+amygdala+pathology+in+alpha-synuclein+transgenic+mice.">Age-dependent cognitive decline and amygdala pathology in alpha-synuclein transgenic mice.</a> Neurobiol Aging. 28, 1421-1435 (2007).</li><li>Fleming, S. M., Fernagut, P. O., Chesselet, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+of+parkinsonism:+strengths+and+limitations.">Genetic mouse models of parkinsonism: strengths and limitations.</a> NeuroRx. 2, 495-503 (2005).</li><li>Kahle, P. J., 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=Selective+insolubility+of+alpha-synuclein+in+human+Lewy+body+diseases+is+recapitulated+in+a+transgenic+mouse+model.">Selective insolubility of alpha-synuclein in human Lewy body diseases is recapitulated in a transgenic mouse model.</a> Am J Pathol. 159, 2215-2225 (2001).</li><li>Chesselet, M. F., Richter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+Parkinson's+disease+in+mice.">Modelling of Parkinson's disease in mice.</a> Lancet Neurol. 10, 1108-1118 (2011).</li><li>Deroose, C. M., Reumers, V., Debyser, Z., Baekelandt, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+genes+at+work+in+the+living+brain+with+non-invasive+molecular+imaging.">Seeing genes at work in the living brain with non-invasive molecular imaging.</a> Curr Gene Ther. 9, 212-238 (2009).</li><li>Manfredsson, F. P., 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=rAAV-mediated+nigral+human+parkin+over-expression+partially+ameliorates+motor+deficits+via+enhanced+dopamine+neurotransmission+in+a+rat+model+of+Parkinson's+disease.">rAAV-mediated nigral human parkin over-expression partially ameliorates motor deficits via enhanced dopamine neurotransmission in a rat model of Parkinson's disease.</a> Exp Neurol. 207, 289-301 (2007).</li><li>Vercammen, L., 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=Parkin+protects+against+neurotoxicity+in+the+6-hydroxydopamine+rat+model+for+Parkinson's+disease.">Parkin protects against neurotoxicity in the 6-hydroxydopamine rat model for Parkinson's disease.</a> Mol Ther. 14, 716-723 (2006).</li><li>Winklhofer, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parkin+protein+as+a+therapeutic+target+in+Parkinson's+disease.">The parkin protein as a therapeutic target in Parkinson's disease.</a> Expert opinion on therapeutic targets. 11, 1543-1552 (2007).</li><li>Kirik, D., 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=Parkinson-like+neurodegeneration+induced+by+targeted+overexpression+of+alpha-synuclein+in+the+nigrostriatal+system.">Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system.</a> J Neurosci. 22, 2780-2791 (2002).</li><li>Kirik, D., 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=Nigrostriatal+alpha-synucleinopathy+induced+by+viral+vector-mediated+overexpression+of+human+alpha-synuclein:+a+new+primate+model+of+Parkinson's+disease.">Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson's disease.</a> Proc Natl Acad Sci U S A. 100, 2884-2889 (2003).</li><li>Lauwers, E., 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=Neuropathology+and+neurodegeneration+in+rodent+brain+induced+by+lentiviral+vector-mediated+overexpression+of+alpha-synuclein.">Neuropathology and neurodegeneration in rodent brain induced by lentiviral vector-mediated overexpression of alpha-synuclein.</a> Brain pathology. 13, 364-372 (2003).</li><li>Klein, R. L., King, M. A., Hamby, M. E., Meyer, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+cell+loss+induced+by+human+A30P+alpha-synuclein+gene+transfer+to+the+rat+substantia+nigra.">Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra.</a> Hum Gene Ther. 13, 605-612 (2002).</li><li>Vander Perren, A., Van den Haute, C., Baekelandt, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Vector-Based+Models+of+Parkinson's+Disease.">Viral Vector-Based Models of Parkinson's Disease.</a> Curr Top Beh Neurosci. , (2014).</li><li>Van der Perren, A., 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=Longitudinal+follow-up+and+characterization+of+a+robust+rat+model+for+Parkinson's+disease+based+on+overexpression+of+alpha-synuclein+with+adeno-associated+viral+vectors.">Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors.</a> Neurobiol Aging. , (2014).</li><li>Van der Perren, A., 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=Efficient+and+stable+transduction+of+dopaminergic+neurons+in+rat+substantia+nigra+by+rAAV++2/1,+2/2,+2/5,+2/6.2,+2/7,+2/8+and+2/9.">Efficient and stable transduction of dopaminergic neurons in rat substantia nigra by rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9.</a> Gene Ther. , (2011).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Soueid, J., Nokkari, A., Makoukji, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+and+Methods+of+Animal+Brain+Surgery:+Perfusion,+Brain+Removal,+and+Histological+Techniques.+Brain+Neurotrauma:+Molecular,+Neuropsychological,+and+Rehabilitation+Aspects.">Techniques and Methods of Animal Brain Surgery: Perfusion, Brain Removal, and Histological Techniques. Brain Neurotrauma: Molecular, Neuropsychological, and Rehabilitation Aspects.</a> Frontiers in Neuroengineering. , (2015).</li><li>Dale, G. E., 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=Relationships+between+Lewy+bodies+and+pale+bodies+in+Parkinson's+disease.">Relationships between Lewy bodies and pale bodies in Parkinson's disease.</a> Acta Neuropathol. 83, 525-529 (1992).</li><li>Dawson, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+flies+and+mice.">Neurobiology of flies and mice.</a> Science. 288, 631-632 (2000).</li><li>Dawson, T., Mandir, A., Lee, M. <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+PD:+pieces+of+the+same+puzzle?.">Animal models of PD: pieces of the same puzzle?.</a> Neuron. 35, 219-222 (2002).</li><li>LeVine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+beta-sheet+amyloid+fibril+structures+with+thioflavin+T.">Quantification of beta-sheet amyloid fibril structures with thioflavin T.</a> Methods Enzymol. 309, 274-284 (1999).</li><li>Oliveras-Salva, M., 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=rAAV2/7+vector-mediated+overexpression+of+alpha-synuclein+in+mouse+substantia+nigra+induces+protein+aggregation+and+progressive+dose-dependent+neurodegeneration.">rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration.</a> Mol Neurodegener. 8, (2013).</li></ol>

The authors declare that there is no actual or potential conflict of interest.

There are several critical steps within the protocol. The vector titer as well as the vector purity directly influences the phenotypic outcome of the model. Excessive vector titers or insufficiently purified vector batches may result in non-specific toxicity. Therefore, the use of high quality vector batches and appropriate control vectors is indispensable. Further, the exact positioning of the rat&#39;s head in the stereotaxic frame and the accurate determination of the coordinates is essential in targeting the substant...

To study the pathophysiology of PD and to develop novel therapeutic strategies, there is an urgent need for animal models that closely resemble the neuropathology, physiology and motor symptoms of human PD. The higher the predictive value, the better we can translate new therapies from animal models to patients.
The discovery of alpha-synuclein (&#945;-SYN) as the first PARK gene in 1997 led to the development of the first genetic PD models. Many transgenic mice overexpressing human wild-type (WT) or mutant (A30P, A53T) &#945;-SYN have been generated over the last decade. The levels of &#945;-SYN overexpression have proven to be crucial in the development of the pathology. Also the mouse strain, the presence or absence of endogenous &#945;-SYN and whether the full length or a truncated form is expressed, plays a role (detailed review by Magen and Chesselet1). Overexpression of both WT and several clinical mutants of human &#945;-SYN in transgenic mice induces pathological accumulation of &#945;-SYN and neuronal dysfunction2-6. However, until now most transgenic &#945;-SYN mouse models failed to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits.
This hurdle was overcome by direct targeting of the substantia nigra (SN) with viral vectors overexpressing &#945;-SYN. Viral vectors are derived from viruses that can easily infect cells, introduce genetic material into their host genome and force the host cell to replicate the viral genome in order to produce new virus particles. Viruses can be engineered to non-replicating viral vectors that retain their ability to enter cells and introduce genes. By deleting parts of the viral genome and replacing them by the genes of interest, application of the vector will result in a single round infection without replication in the host cell, generally designated as &#39;transduction&#39;. Viral vectors can be used for both overexpression and gene silencing. The expressed transgene can be a reporter protein (e.g. green fluorescent protein or firefly luciferase)7, a therapeutic protein for gene therapy applications8-10 or, as we will focus on in this paper, a disease-related protein used for disease modeling11-14.
Viral vector-mediated gene delivery provides an alternative way to express transgenes in the CNS with several advantages. Using local transgene delivery, specific brain regions can be targeted. Further, transgene expression can be induced during adulthood decreasing the risk of compensatory mechanisms during development. Also, models can be created in different species and strains. And finally, different transgenes can easily be combined. Using viral vectors, high transgene expression levels can be achieved, which might be crucial since the disease onset and severity frequently depend on the level of overexpression.
Several vector systems based on different viruses have been developed. The choice of the vector system depends on the size of the gene of interest, the required duration of gene expression, the target cell and biosafety issues. For stable gene transfer in the brain, lentiviral (LV) and recombinant adeno-associated viral (rAAV) vectors are now considered the vector systems of choice since they lead to efficient and long-term gene expression in the rodent brain. For specific targeting of the dopaminergic neurons (DN) of the SN, rAAV vectors have gradually outcompeted LV vectors because of their higher titers and transduction efficiency of DN.
The best &#945;-SYN based rodent models currently available have been developed from a combined approach using newer AAV serotypes (rAAV 1, 5, 6, 7, 8) and optimized vector constructs, titers, and purity15,16. The vector titer as well as the vector purity directly influences the phenotypic outcome of the model. Excessive vector titers or insufficiently purified vector batches may result in non-specific toxicity. Therefore, appropriate control vectors are indispensable. Considerable time investment in the viral vector production, upscaling, and purification procedures have also proven essential to obtain reproducible and high quality vector batches.

<table border="0" cellpadding="0" cellspacing="0" width="1484">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:391px;">Female 8 weeks old Wistar rats</td>
			<td style="width:227px;">Janvier</td>
			<td style="width:187px;">/</td>
			<td style="width:681px;">200-250 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine (Nimatek)</td>
			<td>Eurovet animal health</td>
			<td>804132</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medetomidine (Dormitor)</td>
			<td>Orion-Pharma/ Janssen Animal Health</td>
			<td>1070-499</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Local anesthetic for scalp and ears: Xyloca&#239;ne 2% gel</td>
			<td>Astrazeneca</td>
			<td>0137-547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Terramycine</td>
			<td>Pfizer</td>
			<td>0132-472</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bupr&#233;norphine (Vetergesic)</td>
			<td>Ecuphar</td>
			<td>2623-627</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Jodium 1% isopropanol</td>
			<td>VWR</td>
			<td>0484-0100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stereotactic head frame</td>
			<td>Stoeling</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hamilton Syringe (30 gauge -20mm -pst 2)</td>
			<td>Hamilton/ Filter Service</td>
			<td>7803-07</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">atipamezole (Antisedan)</td>
			<td>Orion-Pharma/Elanco</td>
			<td>1300-185</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rAAV A53T &#945;-SYN vector</td>
			<td>LVVC, KU Leuven</td>
			<td>/</td>
			<td>https://gbiomed.kuleuven.be/english/research/50000715/laboratory-of-molecular-virology-and-gene-therapy/lvvc/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium pentobarbital (Nembutal)</td>
			<td>Ceva Sant&#233;</td>
			<td>0059-444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microtome</td>
			<td>Microm</td>
			<td>HM650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit polyclonal synuclein Ab</td>
			<td>Chemicon</td>
			<td>5038</td>
			<td>1:5000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit polyclonal TH Ab</td>
			<td>Chemicon</td>
			<td>152</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lutetium oxyorthosilicate detector-based FOCUS 220 tomograph</td>
			<td>Siemens/ Concorde Microsystems</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">radioligand: 18F-FECT</td>
			<td>In house</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-dopa: Prolopa 125</td>
			<td>Roche</td>
			<td></td>
			<td>6mg/kg i.p.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, Glutamax</td>
			<td>Life Technologies</td>
			<td>N&#176; 31331-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Foetal bovine serum</td>
			<td>Life Technologies</td>
			<td>N&#176; 10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 kD linear polyethylenimine (PEI)</td>
			<td>Polysciences</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OptiPrep Density Gradient Medium: Iodixanol</td>
			<td>Sigma</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optimen</td>
			<td>Life Technologies</td>
			<td>N&#176; 51985-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paxinos 1 watston steretactic atlas, fourth Edition</td>
			<td>Elsevier</td>
			<td>/</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an alpha-synuclein based rat model for parkinson's disease via stereotactic injection of a recombinant adeno-associated viral vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

The overall scheme of the experiment is depicted in Figure 1
rAAV 2/7-mediated overexpression of A53T &#945;-SYN induces dopamine-dependent motor deficits. 
To examine whether the level of &#945;-SYN overexpression is sufficient to induce motor impairments in the rats, we subjected the rats to the cylinder test to evaluate spontaneous forelimb use (Figure 3A). From 3 weeks ...

Watch this Scientific Journal Video about Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector at JoVE.co

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

The overall goal of this procedure is to develop a viral vector based animal model for Parkinson's Disease via Stereotactic Injection in the central nervous system. This method can be used to develop normal animal models which allow for pre-clinical drug testing and can be beneficial in studying the molecular mechanism of Parkinson's Disease as well as many other neurodegenerative disorders. The main advantage of this technique is that it can be used for specific targeting of brain regions.High trenching brain expression can be achieved and that it can be used to create animal models in specific animal strains and species. The different final vector systems have been developed. The choice of the vector system depends on the size of the trenching you want to express, the duration of the gene expression, the sounds that you want to target, and bio-safety issues.Demonstrating the procedure will be Annelies Aertgeerts a technician from our laboratory. Begin by properly anesthetizing an eight week old female Wistar rat according to approved protocols. Check that a surgical plane of anesthesia has been achieved by squeezing each paw, and noting an absence of the withdrawal reflex.Next, use a MicroTransponder implanter to place a MicroTransponder on the back of the rat. Check that the MicroTransponder is positioned correctly and that a readout can be obtained. Now cut the hair on the scalp of the rat, and apply local anesthetic to the scalp and ears.Transfer the rat to a laminar flow hood, and perform the rest of the procedure using aseptic technique. Place the rat in the stereotaxic frame, by securing the ear bars, followed by the mouth and nose bar. Cover the body of the rat with a paper blanket to avoid a drop in body temperature.Apply an ocular lubricant to prevent the eyes from drying. Next disinfect the scalp according to approved procedures. One percent iodine in 70%isopropyl is used here.After making a small incision in the mid-line of the scalp, gently scrape away the membranes on the skull, and rinse with saline. After allowing the skull to dry, ensure that bregma and lambda can clearly be seen. Now, fill a 10 micro-liter, 30 gauge 20 milometer microinjection-syringe with recombinant AAV and place it onto the motorized micro-injection pump connected to the stereotaxic instrument.For specific transaction of the Dopaminergic neurons of the substantia nigra, recombinant AV vectors are the first choice because of their high titers and efficiency for transducing Dopaminergic neurons. Test the flow, by releasing a drop of AAV. Dispose of any released AAV in a polyvalent cleaning detergent.Visually check that the head is fixed straight in the head frame. Then check that the skull is flat, by first moving the tip of the needle to bregma and measuring the height at this point, by lowering the tip of the needle in the dorsal-ventral direction until in touches the skull. Repeat the procedure at lambda.After returning the needle to bregma, move the needle in the anterior posterior and medial lateral direction to the stereotaxic coordinates for microinjection. Write down the coordinates. At the site of injection, measure the height of the skull as before, and ensure that it does not differ from more than 0.3mm from the height of bregma.Then carefully drill a two milimeter hole in the skull. Once the hole is drilled, measure the height of the dura to determine the reference from which to apply the dorsal-ventral coordinate. Penetrate the dura using a 26 gauge needle.Absorb any blood with the sterile tissue, and proceed only after all bleeding has stopped. Now slowly lower the needle of the pre-loaded microinjection syringe into the brain to the dorsal-ventral coordinate. And pause for one minute.Then inject three micro-liters of AAV at a rate of 0.25 micro-liters per minute. After the injection, allow the needle to remain in place for another five minutes to prevent back flow along the needle track before slowly removing it. Stitch the scalp using coded braided polyester 3.0 and disinfect the skin with one percent iodine and 70%isopropyl.To loosen the nose and mouth bar, and then the two ear bars, to gently remove the animal from the stereotaxic instrument. At this time administer analgesia, and if required, an anesthesia reversal agent. Then place the rat in a clean cage on a heating plate set to 38 degrees Celsius, and monitor closely until it wakes up.Following surgery, the kinetics of neurodegeneration can be studied in the whole animal using behavioral tests and PET imaging and after sacrifice, using immunohistochemical techniques. The cylinder test to evaluate spontaneous forelimb use, was performed at various time points following microinjection of recombinant AAV expressing A53T Mutation alpha synuclein into the substantia nigra. From three weeks after injection, the significant motor impairment was seen in rats that received the median dose.At four weeks after injection, a 50%decrease in spontaneous contralateral forepaw use was observed. Whereas the control eGFP injected animals showed no asymmetry in forepaw use. Rats that received a higher dose of recombinant AAV 2/7A53T alpha synuclein, showed a more pronounced impairment of forepaw use at 29 days after injection.To prove that the observed motor impairment was dopamine dependent, a single dose of L-doba was administered. When the cylinder test was repeated 45 minutes after L-dopa treatment, a full recovery of the forepaw use in the recombinant AAV 2/7A53T alpha synuclein injected animals, was observed. To follow up the kinetics of nigrostriatal dopaminergic neurodegeneration non-invasively over time, in individual animals, dopamine transported binding was quantified using small-animal positron emission tomography with F-18FECT as the radioligand.DAT binding significantly decreased in the ipsilateral caudate budiman of recombinant AAV 2/7A53T alpha synuclein injected rats over time. After 32 days, a decrease in DAT binding of up to 85%was observed. As the positive control, injection of the neurotoxin 6 OHDA in the SN induced 90%loss of DAT binding within seven days.Brain sections from rats microinjected with recombinant AAV 2/7A53T alpha synuclein over time were immunostained for tyrosine hydroxylase a marker of dopaminergic neurons. This figure demonstrates the progressive loss of dopaminergic neurons over a 29 day period after microinjection. Once mastered, this technique can be done in 45 minutes.While attempting this procedure, it is important to use high quality final vector matches. Following this procedure, other techniques, like non-invasive PET imaging, behavior analysis, and Immunohistochemical analysis can be performed in order to determine the level of neurodegeneration and neuropathology. After watching this video, you should have a good idea of how to perform a viral vector injection into the substantia nigra in order to develop viral vector based animal models for Parkinson's Disease.Don't forget that working with viral vectors can be hazardous, and that precautions such as working under a laminar flow and proper waste decontamination should always be taken following this procedure.

development-an-alpha-synuclein-based-rat-model-for-parkinson-s

Research

JoVE Journal

Medicine

13.1K visualizações.  KU Leuven. The overall goal of this procedure is to develop a viral vector based animal model for Parkinson's Disease via Stereotactic Injection in the central nervous system. This method can be used to develop normal animal models which allow for pre-clinical drug testing and can be beneficial in studying the molecular mechanism of Parkinson's Disease as well as many other neurodegenerative disorders. The main advantage of this technique is that it can be used for specific targeting of brain regions.

Vídeo: Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector