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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; &#160;&#160;
1. Recombinant Adeno-Associated Virus (rAAV)&#160;Production and Purification
Note: rAAV vector production and purification were performed by the Leuven Viral Vector Core (LVVC).
<ol>
	<li>Briefly, transfect subconfluent low (&#60;50) passage adherent HEK 293T cells using a 25kD linear polyethylenimine 150 nM NaCl transfection solution and three different plasmids in a ratio of 1:1:1 in DMEM medium 2% fetal bovine serum. After 24 hr of incubation at 37 &#176;C in a 5% CO2, replace the medium with fresh DMEM medium 2% fetal bovine serum. 
	Note: The plasmids include the constructs for the AAV7 serotype, the AAV transfer plasmid encoding the human A53T mutant alpha-synuclein (&#945;-SYN) under the control of the CMVie enhanced synapsin1 promoter, and the pAdvDeltaF6 adenoviral helper plasmid.</li>
	<li>Harvest the medium 5 days after transient transfection and concentrate using tangential flow filtration.</li>
	<li>Purify the rAAV vector particles from the concentrated medium using an iodixanol step gradient.</li>
	<li>Use standard techniques of real-time PCR for genomic copy (GC) determination. In this protocol, a vector titer of 3.0 E11 GC/ml was used to develop an &#945;-SYN based rat model for Parkinson&#39;s disease (PD).</li>
</ol>
2. Stereotactic injection of rAAV &#945;-SYN Vector in the substantia nigra (SN) of the Rat (Figure 1)
<ol>
	<li>House eight-week-old female Wistar rats weighing about 200-250 g under a normal 12 hr light/dark cycle with free access to pelleted food and tap water.</li>
	<li>Submit the rat to intraperitoneal (i.p.) anesthesia containing a mixture of ketamine (60 mg/kg) and medetomidine (0.4 mg/kg). Once the rat is anesthetized and doesn&#39;t react when squeezing the different paws, administer a micro-transponder subcutaneously on the back of the rat for further recognition using a micro-transponder implanter. Check if the micro-transponder is positioned correctly and can be read out by the reading device.</li>
	<li>Cut the hair on top of the scalp. Apply a local anesthetic on both the scalp and the ears. Perform the rest of the surgical procedure under a laminar flow using aseptic techniques.</li>
	<li>Place the rats in a stereotactic head frame using two ear bars, a mouth, and a 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.</li>
	<li>Disinfect the scalp with jodium 1% in isopropanol 70% and make a small incision in the midline of the scalp. Gently scrape away the membranes on the skull and rinse with saline. Let the skull dry for several minutes. Observe the cranial sutures and the two reference points: Bregma and Lambda.</li>
	<li>To inject the rAAV vector into the SN, define the coordinates towards Bregma (anteroposterior: 5.3 mm; mediolateral: 2.0 mm; and dorsoventral: 7.2 mm calculated from the dura). 
	Note: The three-dimensional coordinates for each region of interest can be calculated using a stereotaxic atlas of the rat brain, applying Bregma as anatomical reference point.</li>
	<li>Fill a 10 &#181;l microinjection syringe (30-gauge 20 mm) with the rAAV vector and place it in the stereotaxic instrument connected to a motorized microinjection pump. Control the volume by releasing a drop of vector and eliminate in a polyvalent cleaning detergent pH 9 (e.g.&#160;RBS).</li>
	<li>Visually check if the head is fixed straight in the head frame and evaluate the left-right axis. Carefully visually define the anteroposterior and mediolateral coordinates for Bregma and Lambda and measure their height using a 30-gauge 20 mm needle in the dorsoventral arm of the stereotactic frame.
	<ol>
		<li>Allow a maximum of 0.3 mm difference in height between Bregma and Lambda. Place the needle back on Bregma and apply the anteroposterior and mediolateral coordinates by moving the anteroposterior and the mediolateral arm of the stereotactic frame.</li>
	</ol>
	</li>
	<li>At the place of injection, measure the height of the skull and ensure that it does not differ more than 0.3 mm from the height of Bregma. Drill a hole in the skull with a diameter of approximately 2 mm. Measure the height of the dura, which will serve as a reference for applying the dorsoventral coordinate. Alternatively, subtract a fixed thickness for the skull (0.9 mm).</li>
	<li>Penetrate the dura using a 26-gauge needle and absorb the blood with sterile tissue. Wait until all bleeding has stopped before proceeding.</li>
	<li>Slowly insert the 10 &#181;l microinjection syringe pre-loaded with vector solution into the brain to the pre-determined depth (dorsoventral coordinate). Wait 1 min with the needle in place. Inject 3 &#181;l of vector solution (3.0 E11 genome copies/ml (medium vector dose) or 1.0 E12 GC/ml (high vector dose) of rAAV2/7 &#945;-SYN or eGFP control vector) using the motorized microinjection pump with a throughput of 0.25 &#181;l/min.</li>
	<li>After injection, keep the needle in place for another 5 min before slowly removing it. Stitch the scalp using coated braided polyester 3.0, disinfect with 1% jodium in 70% isopropanol and gently remove the animal from the stereotactic instrument. First loosen the nose and mouth bar, then the two ear bars.</li>
	<li>To reverse the anesthesia, inject the rat intraperitoneally with 0.5 mg/kg atipamezole and place the rat in a clean cage on a heating plate of 38 &#176;C until it wakes up. Cover the rat with a paper blanket to prevent a drop in body temperature.</li>
	<li>Provide easy access to food and water for the first hours. Monitor the rat for the first few days. If necessary, apply analgesia. 
	Note: There is no need to remove the stitches from the skull. After 1-2 weeks the skull is completely repaired, and the stiches come loose.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Female 8 weeks old Wistar rats</td>
			<td>Janvier</td>
			<td></td>
			<td>200-250 g</td>
		</tr>
		<tr>
			<td>Ketamine (Nimatek)</td>
			<td>Eurovet animal health</td>
			<td>804132</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine (Dormitor)</td>
			<td>Orion-Pharma/ Janssen Animal Health</td>
			<td>1070-499</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Local anesthetic for scalp and ears: Xyloca&#239;ne 2% gel</td>
			<td>Astrazeneca</td>
			<td>0137-547</td>
			<td></td>
		</tr>
		<tr>
			<td>Terramycine</td>
			<td>Pfizer</td>
			<td>0132-472</td>
			<td></td>
		</tr>
		<tr>
			<td>Bupr&#233;norphine (Vetergesic)</td>
			<td>Ecuphar</td>
			<td>2623-627</td>
			<td></td>
		</tr>
		<tr>
			<td>Jodium 1% isopropanol</td>
			<td>VWR</td>
			<td>0484-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotactic head frame</td>
			<td>Stoeling</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringe (30 gauge -20mm -pst 2)</td>
			<td>Hamilton/ Filter Service</td>
			<td>7803-07</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole (Antisedan)</td>
			<td>Orion-Pharma/Elanco</td>
			<td>1300-185</td>
			<td></td>
		</tr>
		<tr>
			<td>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>
			<td>DMEM, Glutamax</td>
			<td>Life Technologies</td>
			<td>N&#176; 31331-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>N&#176; 10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>25 kD linear polyethylenimine (PEI)</td>
			<td>Polysciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium: Iodixanol</td>
			<td>Sigma</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimen</td>
			<td>Life Technologies</td>
			<td>N&#176; 51985-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Paxinos 1 watston steretactic atlas, fourth Edition</td>
			<td>Elsevier</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a viral vector injection into the rat substantia nigra for neurodegenerative disease modeling

Source: Van der Perren, A. et al., <a href="https://app.jove.com/v/53670">Development of an Alpha-synuclein Based Rat Model for Parkinson&#39;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector.</a>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the process of injecting a viral vector into the rat substantia nigra to model Parkinson&#39;s disease. It outlines the steps for preparing the rat, microinjecting the viral vector into the substantia nigra, and the recovery procedure.

<img alt="Figure 1" src="/files/ftp_upload/23088/23088_Figure1.jpg" /> 
Figure 1: Stereotactic injection of rAAV2/7 vector encoding &#945;-SYN in the SN of the rat. (A,&#160;B,&#160;E) The cranial sutures on the rat skull, defining the two reference points: Bregma and Lambda. (C) A stereotaxic atlas of the rat brain presenting the region of injection namely the SN. (D) A Wistar rat p...

A Viral Vector Injection into the Rat Substantia Nigra for Neurodegenerative Disease Modeling

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; ...

Secure an anesthetized rat in a stereotactic frame.
Disinfect the scalp and make a cranial midline incision to expose the skull.&#160;
Identify the bregma and lambda, two reference points on the skull, to determine the injection site.
Prepare a microinjection syringe containing a viral vector suspension.
The viral vector carries a transgene encoding alpha-synuclein, a protein that undergoes misfolding and aggregation in the neurons of the substantia nigra in neurodegenerative diseases.
Position the syringe in the stereotaxic instrument connected to a microinjection pump.
Create a burr hole at the injection site, avoiding brain injury.
Penetrate the dura with a needle and clean any blood with sterile tissue.
Insert the microinjection needle through the burr hole to reach the substantia nigra.
Slowly inject the viral suspension, ensuring even distribution.
Pause briefly to prevent backflow, then slowly withdraw the needle.
Suture the incision.
The model is now ready for further analysis.

a-viral-vector-injection-into-rat-substantia-nigra-for

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

73 Görüntüleme. Kaynak: Van der Perren, A. et al., Rekombinant Adeno ile İlişkili Viral Vektörün Stereotaktik Enjeksiyonu Yoluyla Parkinson Hastalığı için Alfa-sinüklein Bazlı Sıçan Modelinin Geliştirilmesi.J. Vis. Uzm.(2016). Bu video, Parkinson hastalığını modellemek için sıçan substantia nigra'ya viral bir vektör enjekte etme sürecini göstermektedir. Sıçanın hazırlanması, viral vektörün substantia nigra'ya mikroenjekte edilmesi ve kurtarma prosedürü için adımları ana hatla...

Video: Nörodejeneratif Hastalık Modellemesi için Sıçan Substantia Nigra'ya Viral Vektör Enjeksiyonu - Kavram

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Kemirgen Beyin Mikroenjeksiyon Moleküler Alt Tabakalar Motive Davranış Eğitim için

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

JoVE Journal

Davranış

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Eşzamanlı Elektrofizyolojik Kayıt ve kemirgen Beyindeki inhibitör Ajanların Mikro-enjeksiyon

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Nörobilim

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Aracı Olarak Adeno-ilişkili virüs kullanarak Hastalığında Retina Engelleri Eğitim için

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Viral Vector Injection into the Rat Substantia Nigra for Neurodegenerative Disease Modeling

TRANSKRIPT

A Viral Vector Injection into the Rat Substantia Nigra for Neurodegenerative Disease Modeling