Name: digital droplet pcr method for the quantification of aav transduction efficiency in murine retina
Uploaded: 2021-12-25T14:00:00
Description: Watch this Scientific Journal Video about Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina at JoVE.com

We would like to thank Oezkan Keles, Josephine J&#252;ttner, and Prof. Botond Roska, Institute of Molecular and Clinical Ophthalmology Basel, Complex Viruses Platform for their help and support for AAV production. We also would like to thank Prof. Jean Bennett, Perelman School of Medicine, the University of Pennsylvania for the AAV8/BP2 strain. Animal work is performed at the Gebze Technical University animal facility. For that, we thank Leyla Dikmetas and Prof. Uygar Halis Tazebay for technical assistance and support for animal husbandry. We also would like to thank Dr. Fatma Ozdemir for her comments on the manuscript. This work is supported by TUBITAK, grant numbers 118C226 and 121N275, and Sabanci University Integration grant.

All experimental protocols were accepted by the Sabanci University ethics committee and experiments were conducted in accordance with the statement of &#39;The Association for Research in Vision and Ophthalmology&#39; for the use of animals in research
1. Small scale AAV production12
<ol>
	<li>Culture HEK293T cells using 15 cm plates in complete 10 mL of DMEM/10% FBS until 70-80% confluency.</li>
	<li>Prepare the transfection mixture with 20 &#181;g o...

<ol>
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In this protocol, we generated two AAV vectors that have different capsid proteins and then titered them accordingly. One of the most crucial steps of this protocol is to produce sufficient amounts of AAVs that will yield detectable reporter expression after the transduction12,13. 
Titering of AAVs is also an important factor to adjust dosages of AAV for intravitreal injections. Once these important criteria are achieved, it is feasi...

Adeno associated viruses (AAVs) are now commonly used for a variety of retinal gene therapy studies. AAVs provide a safe and efficient way of gene delivery with less immunogenicity and fewer genome integrations. AAV entry into the target cell occurs through endocytosis, which requires binding of receptors and co-receptors on the cell surface1,2. Therefore, the transduction efficiency of AAVs for different cell types depends mainly on the capsid and its interactions with the host cell receptors. AAVs have serotypes and each serotype can have distinct cellular/tissue tropisms and transduction efficiencies. There are also artificial AAV serotypes and variants generated by chemical modification of the virus capsid, production of hybrid capsids, peptide insertion, capsid shuffling, directed evolution, and rational mutagenesis3. Even minor changes in amino acid sequence or capsid structure can have an influence on interactions with host cell factors and result in different tropisms4. In addition to capsid variants, other factors like injection route and batch-to-batch variation of AAV production can affect the transduction efficiency of AAVs in the neuronal retina. Therefore, reliable methods for the comparison of transduction rates for different variants are necessary.
The majority of the methods for determining AAV transduction efficiency rely on reporter gene expression. These include fluorescent imaging, immunohistochemistry, western blot, or histochemical analysis of the reporter gene product5,6,7. However, due to the size constraint of AAVs, it is not always feasible to include reporter genes to monitor the transduction efficiency. Using strong promoters like the hybrid CMV enhancer/chicken beta-actin or Woodchuck hepatitis post-transcriptional regulatory element (WPRE) as an mRNA stabilizer sequence further complicates the size problem8. Therefore, it will also be beneficial to define the transduction rate of injected AAVs with a more direct methodology.
Digital droplet PCR (dd-PCR) is a powerful technique to quantify target DNA from minute amounts of samples. dd-PCR technology depends on encapsulation of the target DNA and PCR reaction mixture by oil droplets. Each dd-PCR reaction contains thousands of droplets. Each droplet is processed and analyzed as an independent PCR reaction9. Analysis of droplets enables calculating the absolute copy number of target DNA molecules in any sample by simply using the Poisson algorithm. Since the transduction efficiency of the AAVs is correlated with the copy number of AAV genomes in the neuronal retina, we used the dd-PCR method to quantify AAV genomes.
Here, we describe a dd-PCR methodology to calculate the transduction efficiency of AAV vectors from retinal genomic DNA6,10. First, AAVs that express tdTomato reporter were generated using the small scale protocol, and titered by the dd-PCR method11. Secondly, AAVs were intravitreally injected into the neuronal retina. To demonstrate the transduction efficiency, we first quantified tdTomato expression using fluorescent microscopy and ImageJ software. This was followed by the isolation of genomic DNA for the quantification of AAV genomes in injected retinas using dd-PCR. Comparison of tdTomato expression levels with the transduced AAV genomes quantified by the dd-PCR showed that the dd-PCR method accurately quantified the transduction efficiency of AAV vectors. Our protocols demonstrated a detailed description of a dd-PCR based methodology to quantify AAV transduction efficiencies. In this protocol, we also show the absolute number of AAV genomes that are transduced after intravitreal injections by simply using the dilution factor after genomic DNA isolation and the dd-PCR results. Overall, this protocol provides a powerful method, which would be an alternative to reporter expression to quantify transduction efficiencies of AAV vectors in the retina.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-Well Semi-Skirted ddPCR plates</td>
			<td>BioRad</td>
			<td>12001925</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Amicon Filter</td>
			<td>Millipore</td>
			<td>UFC910096</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>C1000 TOUCH 96 DEEP WELLS</td>
			<td>BioRad</td>
			<td>1851197</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>C57BL/6JRj mice strain</td>
			<td>Janvier</td>
			<td>C57BL/6JRj</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>DG8 gaskets</td>
			<td>BioRad</td>
			<td>1863009</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>DG8 Cartridges</td>
			<td>BioRad</td>
			<td>1864008</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604Q</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>PAN BIOTECH</td>
			<td>L 1825</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Droplet generation oil eva green</td>
			<td>BioRad</td>
			<td>1864006</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Droplet reader oil</td>
			<td>BioRad</td>
			<td>1863004</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAN BIOTECH</td>
			<td>p30-3306</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Foil seals for PX1 PCR Plate sealer</td>
			<td>BioRad</td>
			<td>1814040</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD Medical</td>
			<td>320933</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>ADEKA <img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />LA&#199; SANAY<img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" /> VE T<img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />CARET A.<img alt="Equation 1" src="/files/ftp_upload/63038/63038eq01v2.jpg" />.</td>
			<td>N01AB06</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Microinjector MM33</td>
			<td>World Precision Instruments</td>
			<td>82-42-101-0000</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Micron IV</td>
			<td>Phoenix Research Labs</td>
			<td>Micron IV</td>
			<td>Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.</td>
		</tr>
		<tr>
			<td>Mydfrin (%2.5 phenylephrine hydrochloride)</td>
			<td>Alcon</td>
			<td>S01FB01</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Nanofil Syringe 10 &#956;l</td>
			<td>World Precision Instruments</td>
			<td>NANOFIL</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Needle RN G36, 25 mm, PST 2</td>
			<td>World Precision Instruments</td>
			<td>NF36BL-2</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>PEI-MAX</td>
			<td>Polyscience</td>
			<td>24765-1</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>PAN BIOTECH</td>
			<td>P06-07100</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Plasmid pHGT1-Adeno1</td>
			<td>PlasmidFactory</td>
			<td>PF1236</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Pluronic F-68</td>
			<td>Gibco</td>
			<td>24040032</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer system</td>
			<td>BioRad</td>
			<td>1814000</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>QX200 ddPCR EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>1864034</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader/QX200 Droplet Generator</td>
			<td>BioRad</td>
			<td>1864001</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>SPLITTER FORCEP WATCHER 
			MAKER - LENGTH = 13.5 CM</td>
			<td>endostall medical</td>
			<td>EJN-160-0155</td>
			<td>Retina isolation</td>
		</tr>
		<tr>
			<td>Steril Syringe Filter</td>
			<td>AISIMO</td>
			<td>ASF33PS22S</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Tissue Genomic DNA Kit</td>
			<td>EcoSpin</td>
			<td>E1070</td>
			<td>gDNA isolation</td>
		</tr>
		<tr>
			<td>Tobradex (0.3% tobramycin / 0.1% dexamethasone)</td>
			<td>Alcon</td>
			<td>S01CA01</td>
			<td>anti-inflammatory / antibiotic</td>
		</tr>
		<tr>
			<td>Tropamid (% 0.5 tropicamide)</td>
			<td>Bilim <img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />la&#231; Sanayi ve Ticaret A.<img alt="Equation 1" src="/files/ftp_upload/63038/63038eq01v2.jpg" />.</td>
			<td>S01FA06</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Turbonuclease</td>
			<td>Accelagen</td>
			<td>N0103L</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Viscotears (carbomer 2 mg/g)</td>
			<td>Bausch+Lomb</td>
			<td>S01XA20</td>
			<td>lubricant eye drop</td>
		</tr>
	</tbody>
</table>

digital droplet pcr method for the quantification of aav transduction efficiency in murine retina

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

Small scale AAV production is a fast and efficient method that provides vectors for intravitreal injections (Figure 1). Small scale AAV production usually gives titers within the range of 1 x 1012 GC/ml which is sufficient to detect reporter expression in the retina (Figure 2). Titering of AAV using dd-PCR gives consistent results. ITR2 and WPRE specific primers are routinely used and the starting concentration of each target molecule was calculated w...

Watch this Scientific Journal Video about Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina at JoVE.com

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of ...

In this video, we will show how to quantify AAV transduction in mouse retina using digital droplet PCR method, which is extremely sensitive and does absolute quantification of the target DNA. We have developed this method mainly for big AVV constructs that does not allow us to use a reporter gene like CRISPR based constructs. By using this method, we can simply quantify the transduction rate of AAVs in retina.This method relies on sensitivity and absolute quantification of the digital droplet PCR and yields straight forward results. In this video, we will show how to quantify AAV transduction in mouse retina using digital droplet PCR method, which is extremely sensitive and does absolute quantification of the target DNA. We have developed this method mainly for big area constructs that does not allow us to use a reporter gene like CRISPR based constructs.By using this method, we can simply quantify the transduction rate of AAVs in retina. This method relies on sensitivity and absolute quantification of the two droplet PCR and yields straight forward results. Because of the efficiency of the digital droplet PCR methodology, several laboratories already switch from quantitative real-time PCR to digital droplet PCR or AAV titrating.This method can also provide a basis for mitochondrial DNA quantification in retina, as well as checking the efficiency of the CRISPR based mutation correction for gene therapy in the mouse retina. Prepare transfection mixture with AAV helper plasmid, AAV capsid plasmid, AAV vector, and PI solution in five milliliters demand. Add the five milliliters prepared transfection mixture into culture media.Incubate transfected cells at 37 degrees celsius. Collect the media at 48 to 60 hours post transfection. Digest the media with DNAs at a final concentration of 250 units per milliliter for 30 minutes at 37 degrees Celsius and a centrifuge it at 4, 000 g four degrees Celsius for 30 minutes.Pre-wet the regenerated cellulose membrane with PBS. Filter the media with 0.22 micrometers syringe filter into the pre-wetted regenerated cellulose membrane for 100 kilo Daltons. Centrifuge the regenerated cellulose membrane at 4, 000 G four degrees Celsius for 30 minutes and discard the media.Wash and centrifuge the regenerated cellulose membrane with PBS containing 0.001%pluronic F68 three times at 4000 G four degree Celsius for 30 minutes. Collect concentrated AAVs from the top part of the regenerated cellulose membrane and allocate for further use. Here we showed how to make a smaller scale AAV production.After vetting this protocol, you should be able to make AVVs that is sufficient to see the reporter expression in the retina. You can also follow the protocol for digital PCR to be able to titer the produce AAVs. Apply one drop of 0.5%tropicamide and 2.5%phenylephrine hydrochloride containing eye drop before anesthesia to dilate pupils.Anesthetize mice with isoflurane and verify the reflexes. Apply 0.3%tobramycin and 0.1%dexamethasone sterile ophthalmic solution eye drop to each eye before the injection procedure. Use the surgical hook to stabilize the upper eyelid.Slightly pull back to expose the dorsal part of the eye. Tape the hook to the band to hold in position. Remove conjunctiva with the curved-iris scissors to expose the sclera of the eye.Use a fresh and sterile 30G insulin syringe to puncture the sclera. Insert the needle of the micro syringe through the same puncture using micro manipulator. Place the tip of the needle behind the lens in the middle of the eye cup.Inject one micro liter of AAV solution slowly into the vitreous of the eye. Leave the needle in place for one minute and slowly withdraw the needle. Apply anti-inflammatory and anti-bacterial topical gel treatment containing 0.3%tobramycin and 0.1%dexamethasone to eye cup.Apply one drop off 0.5%tropicamide. And 2.5%phenylephrine hydrochloride containing eye drop before anesthesia to dilate pupils. Apply carbamide two milligrams per gram topical gel onto the surface of the eyes.Adjust the mouse stand as needed to center the mouse eye. Perform fundus and fluorescence imaging on both eyes to follow TdTomato expression one week and two weeks after intravitreal injection. Euthanize the animals using carbon dioxide inhalation before the procedure.Shift eye ball forward with the help of a 13.5 centimeters splitter forceps. Remove the lens together with the leftover vitreous. Cut the connection of the eyeball to the optic nerve with a precision curved forceps and gently squeeze retina with the same curved forceps.Isolate genomic DNA with a commercialized proteinase K digest film-based tissue genomic DNA kit. Digest retina at 55 degrees Celsius 200 G for 30 minutes with proteinase K.Lyse retina completely and follow the manufacturer'sprotocol. In these protocols, we have shown intravitreal injection fundus florescence imaging, as well as retina and genomic DNA isolation.These are very critical procedures for retina, and by following this protocol, you should be able to inject the produced AAVs and follow up their expression profile. And ladle that transduction efficiency by quantifying AAV genomes in retinal genomic DNA. Here, you can see the basic application of digital droplet PCR, where you can make an absolute quantification of AAV genome that transduce in the retinal cells.Dilute genomic DNAs from injected retinas in 0.05%pluronic F 68 solution to reach a final concentration of one nanogram per microliter for each sample. Perform droplet generation in a 20 microliters PCR reaction mix, including one nanogram genomic DNA 125 nanomolars primers and 2X evergreen super mix. Load sample mix to the middle part of the cartridge for droplet generation.Afterwards add 70 microliters droplet generation oil into the bottom row of cartridge. Put the gasket on top of cartridge. Make sure that there is no gap between the gasket and the cartridge.Then place it into the droplet generator. Gently take approximately 40 microliters of droplets that are generated in the top bell. Add droplet solution to semi skirted 96-Well PCR reaction plate.Seal the PCR plate with a PCR plate sealer by using aluminum sealing foil. Place the PCR plate into the 96-Well heat sealed thermal cycler. Use the ddPCR protocol indicated in the article.Place PCR plate into the droplet reader to analyze data. After watching this video, you should be able to perform basic digital PCR technique. You can also use exerted cells with minor amounts of material, which is ideal for digital PCR technique.However, you should carefully adjust the target DNA amount to not exceed the limit of digital droplet PCR. Here's the flow chart for the experimental procedure. We performed small-scale AAV production, which is followed by AAV titering.Then we performed intravitreal injection. Afterwards, we quantified the fluorescent intensity by fundus fluorescence imaging. Then isolated the mouse retinas and extracted genomic DNA from retinas for ddPCR.Titering AAV is also critical to be able to correctly quantify AAV transduction. ddPCR depends on generation of droplets that has diluted target AAV genom. Representative ddPCR results shows positive and negative droplets for the AVV genome.Positive above the threshold and negative is below. After multiplying with the dilution factors, titer of AAVs were found to be 10 to the 12 in genome copies per milliliter. In order to correctly quantify AAV tranduction efficiency, several retinas were injected.Injected animals were imaged, and fluorescence mean intensity was calculated. At two weeks time point, retinas were isolated. And ddPCR was performed using WPRE and 18 S primers.Fluorescence intensity of AAV injected retinas were correlated with AAV genome calculations, showing the power of the methodology. Here, we have shown several techniques, including small-scale AAV preparation intravitreal injection.

digital-droplet-pcr-method-for-quantification-aav-transduction

Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Quantification of Immunostained Caspase-9 in Retinal Tissue

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.