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

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

Begin, by properly anesthetizing an eight-week-old female Wistar rat according to approved protocols. Check that a surgical plan 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. 1% iodine in 70% isopropanol is used here. After making a small incision in the midline 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 be clearly seen.
Now, fill a 10 microliter, 30-gauge 20 millimeters microinjection syringe with recombinant AAV, and place it onto the motorized microinjection pump connected to the stereotaxic instrument.
For specific transduction of the dopaminergic neurons of the substantia nigra, recombinant AAV 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 dorsoventral direction, until it touches the skull. Repeat the procedure at lambda.
After returning the needle to bregma, move the needle in the anteroposterior and mediolateral 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 more than 0.3 milimeters from the height of bregma.
Then carefully drill a 2-millimeter hole in the skull. Once the hole is drilled, measure the height of the dura to determine the reference from which to apply the dorsoventral coordinate. Penetrate the dura using a 26-gauge needle. Absorb any blood with a sterile tissue, and proceed only after all bleeding has stopped.
Now slowly lower the needle of the preloaded microinjection syringe into the brain to the dorsal ventral coordinate, and pause for one minute. Then inject three microliters of AAV at a rate of 0.25 microliters per minute. After the injection, allow the needle to remain in place for another five minutes to prevent backflow along the needle track before slowly removing it.
Stitch the scalp using coated braided polyester 3.0, and disinfect the skin with 1% iodine in 70% isopropanol, to loosen the nose and mouth bar, and then the two ear bars to gently remove the animal from the stereotactic 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.

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Neuropathology

Neurodegenerative Diseases

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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>

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...

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.

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

Video: A Viral Vector Injection into the Rat Substantia Nigra for Neurodegenerative Disease Modeling - Experiment

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.

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JoVE Journal

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

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