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

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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.

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	<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>
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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'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.

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

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Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...