Name: ultrahigh resolution mouse optical coherence tomography to aid intraocular injection in retinal gene therapy research
Uploaded: 2018-11-02T12:30:00
Description: Watch this Scientific Journal Video about Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research at JoVE.com

This material is based upon work supported, in part, by the Department of Veterans Affairs (VA), Veterans Health Administration, Office of Research and Development (Biomedical Laboratory Research and Development) (VA Merit Grant 1I01BX000669). JMS is employed, in part, as Staff Physician-Scientist, Ophthalmology, by VA WNY; MCB is employed, in part, by VA WNY. The study was conducted at, and supported in part by, the Veterans Administration Western New York Healthcare System (Buffalo, NY). Contents do not represent the views of the Department of Veterans Affairs or the United States Government. Also supported, in major part, by NIH/NEI R01 grant EY013433 (PI: JMS), NIH/NEI R24 grant EY016662 (UB Vision Infrastructure Center, PI: M Slaughter, Director- Biophotonics Module: JMS), an Unrestricted Grant to the Department of Ophthalmology/University at Buffalo from Research to Prevent Blindness (New York, NY), and a grant from the Oishei Foundation (Buffalo, NY). We acknowledge the gift of the hC1 transgenic RHOP347S&#160;line and the exon 1 mouse RHO knockout from Dr. Janis Lem (Tufts New England Medical Center, Boston, MA), and the gift of the NHR-E transgene model in the heterozygous state on the mouse exon 2 RHO knockout background from Drs. G. Jane Farrar and Peter Humphries (Trinity College, Dublin, IRE).

Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the VA WNY HCS and the University at Buffalo-SUNY. Animals were used according to the stipulations of the Association for Research in Vision and Ophthalmology (ARVO) and the Declaration of Helsinki.
1. Mouse Models
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	<li>Identify mouse models to be evaluated including controls. 
	NOTE: Imaging was performed for a C57BL/6(J), hC1/hC1//mWT/mWT, a partially humanized mous...

<ol>
	<li>Regus-Leidig, H., 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=In-vivo+knockdown+of+Piccolino+disrupts+presynaptic+ribbon+morphology+in+mouse+photoreceptor+synapses.">In-vivo knockdown of Piccolino disrupts presynaptic ribbon morphology in mouse photoreceptor synapses.</a> Front Cell Neurosci. 8 (259), 1-13 (2014).</li><li>Jiang, L., Frederick, J. M., Baehr, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+gene+therapy+in+dominant+retinitis+pigmentosa+and+cone-rod+dystrophy+mouse+models+caused+by+GCAP1+mutations.">RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations.</a> Front Mol Neurosci. 7 (25), 1-8 (2014).</li><li>Seo, S., 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=Subretinal+gene+therapy+of+mice+with+Bardet-Beidl+Syndrome+Type-1.">Subretinal gene therapy of mice with Bardet-Beidl Syndrome Type-1.</a> Invest. Ophthalmol. Vis. Sci. 54 (9), 6118-6132 (2013).</li><li>Molday, L. 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=RD3+gene+delivery+restores+guanylate+cyclase+localization+and+rescues+photoreceptors+in+the+RD3+mouse+model+of+Leber+congenital+amaurosis+12.">RD3 gene delivery restores guanylate cyclase localization and rescues photoreceptors in the RD3 mouse model of Leber congenital amaurosis 12.</a> Hum. Mol. Genet. 22 (19), 3894-3905 (2014).</li><li>Pang, J. 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=AAV-mediated+gene+therapy+in+mouse+models+of+recessive+retinal+degeneration.">AAV-mediated gene therapy in mouse models of recessive retinal degeneration.</a> Curr. Mol. Med. (3), 316-330 (2012).</li><li>Vandenberghe, L. H., Auricchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adeno-associated+viral+vectors+for+retinal+gene+therapies.">Novel adeno-associated viral vectors for retinal gene therapies.</a> Gene Ther. 19 (2), 162-168 (2012).</li><li>Tenenbaum, L., Lehtonen, E., Monahan, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+risks+related+to+the+use+of+adeno-associated+virus-based+vectors.">Evaluation of risks related to the use of adeno-associated virus-based vectors.</a> Curr. Gene Ther. 3 (6), 545-565 (2003).</li><li>Parikh, S., Le, A., Davenport, J., Gorin, M. B., Nusinowitz, S., Matynia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+and+validated+injection+method+for+accessing+the+subretinal+space+via+a+transcleral+posterior+approach.">An alternative and validated injection method for accessing the subretinal space via a transcleral posterior approach.</a> J. Vis. Exp. (118), e54808 (2016).</li><li>Bainbridge, J. W. B., Mistry, A. R., Thrasher, A. J., Ali, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+injection+of+gene+therapy+vectors+and+stem+cells+in+the+perinatal+mouse+eye.">Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye.</a> J. Vis. Exp. (69), e4286 (2012).</li><li>Berger, A., al, , 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=Spectral-domain+optical+coherence+tomography+of+the+rodent+eye:+highlighting+layers+of+the+outer+retina+using+signal+averaging+and+comparison+with+histology.">Spectral-domain optical coherence tomography of the rodent eye: highlighting layers of the outer retina using signal averaging and comparison with histology.</a> PLoSOne. 9 (5), 96494 (2014).</li><li>Muhlfriedel, R., Michalakis, S., Garrido, M. G., Biel, M., Seeliger, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+technique+for+subretinal+injections+in+mice.">Optimized technique for subretinal injections in mice.</a> Methods in Molecular Biology. , (2013).</li><li>Sarra, G. 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=Kinetics+of+transgene+expression+in+mouse+retina+following+subretinal+injection+of+recombinant+adeno-associated+virus.">Kinetics of transgene expression in mouse retina following subretinal injection of recombinant adeno-associated virus.</a> Vision Res. 42, 541-549 (2002).</li><li>Yan, Q. 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HR-SD-OCT provides a simple method for characterization of potential animal models of human disease to determine their usefulness in testing potential therapeutics. The ability to quickly and reliably characterize a potential animal model of human disease is critical to the process of therapeutic drug discovery (e.g., replacement gene therapy, ribozyme or shRNA knockdown gene therapy, combined gene therapy). HR-SD-OCT provides a simple, quick, and non-invasive method for evaluating retinal health that can be use...

Researchers are developing gene therapies for a variety of retinal and retinal degenerative diseases with hopes of translating novel therapeutics into treatments for human disease1,2,3,4,5,6,7,8,9,10,11. Time domain or spectral domain optical coherence tomography (SD-OCT) has been used to investigate the aspects of outer retinal degeneration in specific mouse models of disease12,13,14. HR-SD-OCT has not, however, been extensively used in the context of optimizing evaluation of mouse models to determine the rate and spatial uniformity of retinal degeneration, or in the context of preclinical evaluation of gene based therapeutics, for example, to assess rescue, toxicity, or the spatial extent of vector delivery8,15,16. Once a mouse model is fully characterized, the HR-SD-OCT data can serve as an informative and reliable resource to measure the impact of therapeutics to exert rescue or toxicity in mouse models of retinal degeneration17. Many groups are using subretinal injection as a method of vector delivery due to its efficiency at transducing photoreceptors and retinal pigment epithelium (RPE) cells. However, this remains a difficult method to master, given that it is typically done by free-hand surgery from the corneal surface, and is often fraught with cataracts, bleeding, and unintended retinal detachments occurring simply by manipulation of the posterior vitreous. Many groups still attempt subretinal injections blindly and deliver the virus using manual injections with relatively large diameter stainless steel needles (34G)8,17,18,19,20,21,22, and a few uses optical coherence tomography (OCT) imaging to confirm proper delivery of vector to the retina8,17,20,22. Some improvements in the method have recently been described using microscale needles driven by a micromanipulator22.
We present an integrated approach which aids in the positioning of the needle, and the injections are facilitated by a custom directed stereo ophthalmoscope designed in the lab specifically for visualizing inside the small eye of the mouse17,23. The use of pulled glass micro needles in conjunction with the stereotaxic micromanipulator provide better control of needle placement with no surgical cut down required (i.e., through conjunctivae and connective tissue) prior to injection. The use of the pressure regulated micro injector helps deliver consistent injection volumes, and the injection can be done with much greater stability, precision, and much slower than manual injections performed by a hand-held syringe, thereby decreasing the occurrence of bubble injection into the eye. The smaller needle helps prevent leakage following needle withdrawal because the path is self-sealing. To assess the extent of injection/delivery, many investigative groups rely on finding and assessing the areal extent of enhanced green fluorescence protein (EGFP) expression in the retina (expression construct delivered by the vector) at the experimental end point (euthanasia) to confirm successful injections11,19,20,24. This approach (not utilizing OCT) to verify surgical success wastes an enormous amount of resources in surgical procedural time and animals, since all animals with (unknown) surgical failures need to be maintained, followed with repetitive measures until euthanasia and eye harvest (when EGFP is measured). Confirmation of the location of injection in the retina can be improved using HR-SD-OCT to demonstrate that the injection is located between the correct layers of the retina (i.e. the subretinal space). HR-SD-OCT can also be used to immediately delineate unsuccessful attempts (surgical failures) to identify relevant variables in real surgical time to improve upon the approach. We found that HR-SD-OCT provides numerous advantages in preclinical gene therapy studies by allowing rapid quantitative evaluation of outer retinal degeneration, allowing identification/culling of study animals which do not meet experimental criteria (e.g., incorrect subretinal injection), and to direct follow-up imaging to the region of the eye where vector was delivered (where preclinical effect is most likely) as well as control regions where vector was not delivered. Since its development, the use of SD-OCT has continued to be accepted and used by ophthalmology researchers and is now considered the standard of retinal imaging in retinal scientific studies in mouse or rodent models13,25. HR-SD-OCT and its software capabilities were utilized in unique integrated ways to further the goal of successful quantitative gene therapy in mouse models at every step in the process, including animal model selection, characterization of degeneration in chosen disease models, therapeutic delivery, mapping of vector delivery, and toxicity/efficacy evaluation. The use of HR-SD-OCT allows for more efficient drug discovery at every level of the process. Here we describe these approaches that are used in our RNA Drug Discovery program.

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			<td height="39" style="height:39px;width:251px;">C57BL6 (J)</td>
			<td style="width:245px;">Jackson Laboratories</td>
			<td style="width:215px;">664</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">N129R-</td>
			<td style="width:245px;">N/A</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">2HRho 1T/1T</td>
			<td style="width:245px;">N/A</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="78" style="height:78px;width:251px;">Envisu R-2200 Ocular Coherence Tomography Instrument (OCT)</td>
			<td style="width:245px;">Bioptigen</td>
			<td style="width:215px;">90-R2200-U1-120.&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Retinal Imaging System (RIS)</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Stemi 2000C Microscope</td>
			<td style="width:245px;">Zeiss</td>
			<td style="width:215px;">000000-1106-133</td>
			<td></td>
		</tr>
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			<td height="39" style="height:39px;width:251px;">P-97 Flaming/Brown micropipette puller</td>
			<td style="width:245px;">Sutter Instrument Co</td>
			<td style="width:215px;">p-97</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">MMN-33 micro manipulator&#160;</td>
			<td style="width:245px;">Narishige USA</td>
			<td style="width:215px;">MMN-33</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">PLI-100&#160; micro injector</td>
			<td style="width:245px;">Harvard Apparatus</td>
			<td style="width:215px;">64-1736</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Micropipette Holder (Rotating)</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Micropipette Storage Receptacle</td>
			<td style="width:245px;">World Precision Instruments Inc.</td>
			<td style="width:215px;">E210</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">&#160;Borosilicate glass capillary tubes 1.5-1.8 X 100mm,&#160;</td>
			<td style="width:245px;">Harvard Apparatus</td>
			<td style="width:215px;">30-0053</td>
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			<td height="39" style="height:39px;width:251px;">Cyclopentolate Hydrochloride Ophthalmic Solution, USP&#160; 2%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-097-10</td>
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			<td height="39" style="height:39px;width:251px;">Gentamicin Sulfate Ointment USP, 0.1%</td>
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			<td style="width:245px;">Neslab</td>
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			<td height="39" style="height:39px;width:251px;">Heating plate</td>
			<td style="width:245px;">In House</td>
			<td style="width:215px;">N/A</td>
			<td></td>
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			<td style="width:215px;">N/A</td>
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			<td height="39" style="height:39px;width:251px;">Betadine</td>
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			<td style="width:215px;">BP2818-100</td>
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			<td height="39" style="height:39px;width:251px;">Gloves Nitrile</td>
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			<td style="width:215px;">89038-272</td>
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			<td height="39" style="height:39px;width:251px;">Diagnosys ERG Color Dome instrument</td>
			<td style="width:245px;">Diagnosys Inc.</td>
			<td style="width:215px;">D125</td>
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			<td height="39" style="height:39px;width:251px;">Contact lenses</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
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			<td height="39" style="height:39px;width:251px;">Diagnosys Software</td>
			<td style="width:245px;">Diagnosys Inc.</td>
			<td style="width:215px;">N/A</td>
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			<td height="39" style="height:39px;width:251px;">Origin 6.1 software</td>
			<td style="width:245px;">OriginLab Corp.</td>
			<td style="width:215px;">N/A</td>
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			<td height="39" style="height:39px;width:251px;">Reference electrodes</td>
			<td style="width:245px;">Ocuscience</td>
			<td style="width:215px;">F-Thread Electrode (DTL) 24&#8221;</td>
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ultrahigh resolution mouse optical coherence tomography to aid intraocular injection in retinal gene therapy research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Assessing the Presence, Rate and Uniformity of Model Outer Retinal Degeneration 
Measurements of the ONL were recorded from the OPL to the ELM, defining the limits of the ONL using the caliper tool provided in the instrument software. The goal was to map the progression of outer retinal degeneration in a partially humanized adRP mouse model. Comparable images from a control C57BL/6(J) mouse and an hC1/hC1//mWT/mWT mouse model, expressing two copies of the mutant huma...

Watch this Scientific Journal Video about Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research at JoVE.com

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

The overall goal of this subretinal injection procedure is to deliver a vector to the retina in a minimally invasive and reproducible manner that is observable in real-time. Hello, this method can be used to answer key questions in gene therapy research about vector delivery efficacy and therapeutic agent toxicity. Some advantages of this technique include minimal damage to the retina and other eye structures, precise injection site mapping, and rigorous quantitative in vivo measurements that allow solid statistical inference.For intraocular transgene injection, first place an anesthetized mouse onto the viewing stage of a retinal imaging system microscope and place the tips of a sterile blunt iris forceps at the seven and 10 o'clock positions of the eyelid while pushing the forceps open and down to gently induce proptosis. Use the forceps to coax the eyelids under the eye globe and direct the tip of the needle to about one to 1.5 millimeters below the edge of the corneal limbus. Next, apply a drop of sterile PBS solution to the eye and cover the eye with a sterile cover slip.Carefully drive the needle into the eye through the conjunctiva until a scleral depression is created. Use the micro manipulator to rotate the eye upward until the scleral depression is in focus, then drive the needle forward to create a sharp peak in the retina with the tip of the needle. Using the needle holder, rotate the needle just until the tip of the needle bores through the sclera and the RPE, allowing the flourescein in the tip of the needle to be visible under the retinal tissue in the immediate vicinity of the subretinal space and the retinal pigment epithelium cell monolayer.Drive the tip of the needle downward so it is tangential to the globe, then activate the injection pump with the foot pedal switch. After 0.5 to one microliters of transgene solution has been delivered to the subretinal space, withdraw the needle and confirm that the blood is stable and that fluid does not leak out of the injection tract site. To confirm the success of the injection, record a rectangular volume OCT and compare the images to images of the different kinds of potential injection outcomes.After confirmation of proper injection placement, use that rectangular OCT image to map the extent of the bleb onto the fundus image. To map the extent of the subretinal bleb, open a rectangular volume OCT image of the bleb such that a recognizable landmark within the eye is included in the image. Add as many calipers as necessary to identify the position of the left and right edges of the B scan, as well as the optic nerve head, ONH, if present, and the inflection point where the retina detaches from the RPE and choroid and then save the data.Then compile the saved files and plot the data, effectively creating a map of the injection bleb onto the en face OCT image. Overlay the plotted data onto the fundus image containing the injection site and save the merged image. Use this image to locate regions of interest during follow-up OCT imaging.To assess post injection retinal degeneration, first open a recorded high-resolution spectral domain OCT or HRSD OCT rectangular volume image and select a B scan to be measured. Magnify the selected image until it fills the screen and right click on the image. Click calipers to select the appropriate number of calipers, then use the configure caliper feature to assign the calipers as vertical in the angle block column and turn on the display caliper location to facilitate uniform caliper placement across the retina.Click apply and move each caliper to the appropriate location 0.1 to 0.2 millimeters apart across the B scan, taking care to place one caliper into the center of the optic nerve head when appropriate. When all of the calipers have been placed, click and drag each caliper to the length span of the region of interest, arbitrarily setting calipers not overlaying measurable regions of the B scan to maximum length. To measure the outer nuclear layer thickness, place the top of each caliper at the external limiting membrane and the bottom of each caliper at the bottom of the outer plexiform layer at 0.1 to 0.2 millimeter increments across the retina.After all of the calipers have been placed and adjusted to size, right click on the image and click save caliper data"then repeat the measurements as just demonstrated for every tenth B scan, keeping the calipers open and at the same location on the x-axis and adjust the lengths of the calipers as necessary, without moving them in the x-direction. When all of the scans have been measured, open the saved data files and click date modified"to arrange the files according to time saved. Open all of the files for each B scan measured and compile the raw data from each B scan into a single file from lowest to highest frame number and select the columns of data, including the caliper name, length, and center X columns.Ensure that the center X is the same for all of the B scans measured for each rectangular volume OCT image processed and delete any data from the calipers that were not used to record measurements. Set the caliper located at the center of the optic nerve head to zero and plot the data for X versus multiple Y data sets to get a 3D plot function for the thickness of the outer nuclear layer or any other measured retinal layer. Then compare the outer nuclear layer measurements between the control and experimental animals with corresponding ages to determine the rate and uniformity of any potential retinal degeneration.For 3D mapping of the retinal measurements, review the post-injection OCT images and note any identifiable landmarks, then position the animal to obtain a follow-up SDO CT image from the same region, taking care to include the same identifiable landmarks, and process the recorded images as just demonstrated. It is very important to obtain high-quality OCT images that include identifiable landmarks to facilitate reimaging of the previously injected retinal regions. In order to measure the changes in OS length, calipers were placed from the external limiting membrane to the Brooks membrane, allowing for reliable measurements since these layers are easily identified in OCT images.Differences in the measurements between these layers from different mouse lines were attributed to shorter outer segment lengths. If the subretinal injection is performed successfully, the opening of the subretinal space induces the production of a bleb that can be clearly visualized both in the en face view of the HRSD OCT imager and in the B scan images. Superimposing the border map of the injection site allows segmentation of the retinal tissue measurements and caliper position datasets to allow subsequent statistical testing of the effects of specific therapeutic agents on retinal degeneration.Once mastered, this technique can be completed in ten minutes if performed properly. While attempting this procedure it's important to remember to keep the surgical environment clean and sterile. After its development, this technique paved the way for researchers in the field of gene therapy to monitor the entire subretinal surgical injection process in real-time and created a way to immediately determine surgical success in vivo.Following this injection procedure, OCT and other methods like electro retinography can be performed to answer additional questions about transgene functional rescue and/or toxicity. After watching this video, you should have an understanding how to perform transscleral and transchoroidal subretinal injections, how to map the extent of the injection bleb onto the OCT from this image, and obtain rigorous measurements of the outer retinal layers. Don't forget that working with gene therapy vectors can be hazardous and that precautions, such as wearing the appropriate personal protective equipment should always be used while performing this procedure.

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