Method Article

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

DOI:

10.3791/55894

⸱

November 2nd, 2018

 , 
1Research Service, VA Western New York Healthcare System, 2Department of Ophthalmology, (Ross Eye Institute), Jacobs School of Medicine and Biomedical Sciences, University at Buffalo- SUNY, 3Pharmacology/Toxicology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo- SUNY, 4Physiology/Biophysics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo- SUNY, 5Neuroscience Program, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo- SUNY, 6The RNA Institute, University at Buffalo- SUNY, 7The SUNY Eye Institute

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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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Protocol

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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

  1. Identify mouse models to be evaluated including controls.
    NOTE: Imaging was performed for a C57BL/6(J), hC1/hC1//mWT/mWT, a partially humanized mouse retinal degeneration model homozygous for human mutant RHOP347S hC1 alleles on the wild-type (WT) mouse RHO+/+ genotype26,27, hC1 x BL/6(J), a partially humanized model with a single copy of the mutant human RHOP347S hC1 allele on the WT mouse RHO+/+ genotype (hC1/-//mWT/mWT) (obtained by crossing hC1/hC1//mWT/mWT with C57BL/6(J) mice). The above autosomal dominant retinitis pigmentosa (adRP) models are on the C57BL/6(J) background. A mouse model that is homozygous for two copies of the human WT RHO gene on the mouse RHO knockout background was also used28,29. This line is on the 129Sv background. When this line was crossed with a mouse RHO knockout on the 129Sv background, a single dose of human RHO occurs on the mouse RHO background.
  2. Maintain the animals following the conditions pertinent to the experimental design.
    NOTE: The animals were maintained in the Veterinary Medical Unit (VMU) at the VA WNY HCS. Mice were fed standard lab chow and grown under 12 h:12 h light: dark cycles with soft fluorescent overhead white lights with approximately 300 lux at cage level at approximately 72 ˚F.

2. Mouse Eye Gel

  1. Prepare the optical gel used for retinal imaging30 and surgical procedures.
  2. Combine 2 mg/mL w/v high molecular weight (4 x 106 g/mol carbomer in sterile 1x Phosphate Buffered Saline (PBS).
  3. Mix at room temperature until the gel forms a viscous optically transparent gel.
  4. Transfer the gel into small sterile bottles and centrifuge in a swinging bucket tabletop centrifuge at 350 x g to remove trapped air bubbles.
  5. Apply the gel directly to the cornea to create an interface between the mouse cornea and a premium cover glass (18 mm x 18 mm).

3. HR-SD-OCT Imaging

  1. See the HR-SD-OCT device (Figure 1).
  2. Weigh the animal to determine the proper dose of anesthetic. Then anesthetize the mouse using 25 µL/g of body weight of the 2.5% solution of buffered 2,2,2-tribromoethyl alcohol (Avertin) solution via intraperitoneal injection (IP) and add the eye drops to dilate the pupils after the animal is immobilized.
  3. Confirm that the animal is fully anesthetized by a toe pinch, and be certain the animal does not react.
  4. Trim the whiskers and place the mouse onto the HR-SD-OCT sled.
  5. Position the mouse's eye directly in front of the headpiece lens and manipulate the stage controls until the cornea and iris are located. Keep the cornea hydrated by applying artificial tears.
    NOTE: The fine adjustment micromanipulators on the HR-SD-OCT sled are used to position the mouse such that the pupil aperture is centered and oriented. The optical headpiece is then advanced until the retina becomes visible and the animal is further adjusted to obtain the best possible image.
  6. First, open the software program "Mouse" and click "patient/exam". Second, click "add patient" and enter the pertinent information to identify the animal in the new patient window and "save and Exit". Third, click "add exam" followed by "start exam". Fourth, click "add custom scan" and select "rectangular volume", choose OD or OS for the eye being imaged, then click "add exam". Finally, start aiming the instrument and positioning the animal to obtain the region of interest. After finding the correct region, remove any excess fluid from the eye surface using a sterile cotton tipped applicator just prior to image acquisition in order to further enhance image quality, then click "start snapshot", and if a good image is obtained, click "save scan".
    NOTE: Typical parameters for rectangular HR-SD-OCT images are 900 a-scans/b-scan and 90 b-scans/image. Acquired images are 1.4 mm x 1.4 mm. The region of the retina imaged depends on the specific experiment, but most images are centered on the optic nerve head (ONH).

4. Assessing the Presence, Rate, and Uniformity of Model Outer Retinal Degenerations

  1. Outer Retinal Degeneration Model Evaluation by HR-SD-OCT
    1. Obtain animals with a wide spectrum of ages for both the control and experimental subjects to assess the presence, rate, and uniformity of outer retinal degeneration.
    2. Perform HR-SD-OCT imaging on multiple animals at each age from both cohorts (disease and normal). Use the method described in 3.1.6 to obtain the OCT images.
      NOTE: Data can be collected from a large cohort of animals all at once, which have distributed birthdays spanning a wide period of time (1 year), or a small cohort of animal can be used to collect multiple images over a long period of time (1 year) to obtain similar results.
    3. Open a recorded HR-SD-OCT rectangular volume image, and identify the first b-scan to be measured (ideally will include the ONH or other identifiable landmark) from the ensemble of images. Enlarge the image to fill the screen using the zoom feature in the software.
    4. Open the desired number of calipers by right clicking on the image of the b-scan and then clicking on "Calipers" and finally clicking on as many calipers as desired (they are numbered 1 through 10). Ensure that the calipers show up in lower right corner of the image. Using the "Configure Caliper" feature, assign them all as "vertical" in the angle block column and turn on the "Display Caliper Location" to facilitate uniform placement across the retina and finally, click apply.
      NOTE: The 1st caliper should be placed on the left side of the image when processing the oculus dexter (OD) right eye and the 1st Caliper should be placed on the right side of the image when processing the oculus sinister (OS) left eye images. This results in a nasal to temporal orientation of all the data for both the left and right eyes when plotting.
    5. Using the computer mouse, move each caliper to the desired location (2.0 mm apart) across the b-scan, being sure to place one caliper in the center of the optic nerve head, in b-scan images that include it (set this caliper to zero). Then use the mouse to click and drag the caliper to length to span the region of interest. Arbitrarily set calipers that do not overlay measurable regions of the b-scan to maximum length, and ignore during the data analysis.
    6. Measure the ONL thickness using the Caliper tools, by placing the top of the caliper at the external limiting membrane (ELM) and the bottom of the caliper at the bottom of the outer plexiform layer (OPL). Repeat this for each caliper across the retina at 0.2 mm increments. Save the measurements.
    7. After all calipers have been placed and adjusted to size, right click on the image and click "Save Caliper Data".
    8. Repeat measurements on subsequent b-scans (every 10th scan works well) from the same rectangular volume OCT image spanning the entire retina from inferior to superior regions. Calipers should remain open and at the same location in the X-axis. Adjust the length of the calipers without moving them in the X-direction.
    9. Open the save data files by clicking the small file icon next to the processed b-scan image and click "Go to Data". Click "Date Modified to arrange the files in order based on time saved, and open all the files for each b-scan measured.
    10. Compile the raw data from each b-scan into a single file in order from lowest to highest based on frame number. Select the columns of data including "Caliper name", "Length", and "Center X".
    11. Ensure that the center X is the same for all b-scans measured for each rectangular volume OCT image processed. Delete any data from calipers that were not used to record measurements, and set the caliper located at the center of the optic nerve head to zero.
    12. Plot the data for X vs. multiple Y data sets to get a 3D plot function to plot the overall thickness of the ONL or another measured retinal layer.
    13. Compare ONL measurements between control and experimental animals with corresponding ages to determine the rate and uniformity of any potential retinal degeneration.
    14. Repeat the process for OSL measurements using the same b-scan images. Follow the same methodology used to measure the ONL, except placing the calipers between ELM and Bruch's membrane (BM).
      NOTE: An example of how to place the caliper is shown in Representative Results section (Figure 3B). This should be done on a zoomed in image to decrease error.
    15. Repeat the data analysis for multiple animals for each birthday for both experimental and control cohorts.
  2. Comprehensive measurement and 3D mapping of the ONL or OSL thickness using the software calipers tool
    1. Review the post injection OCT images and make note of any identifiable landmarks, like the ONH or blood vessels in the retina. Then position the mouse to obtain a follow-up SD-OCT from the same region, and be sure to include the same identifiable landmarks.
      NOTE: Ensure that the region of the retina imaged and involved in the retinal detachment is identified in the post injection SD-OCT images. Record a rectangular volume SD-OCT image and save it.
    2. Process the recorded images as described above in steps 4.1.3. to 4.1.14. Save the caliper data and plot as described above.
      NOTE: The resulting array of ONL measurements is used to create a 3D plot depicting the ONL thickness. The position of the calipers along the X-axis allow one to replot the graph placing the optic nerve at the origin (0) along the X-axis. The Y-axis is plotted using the b-scan number and the optic nerve is then used to define the starting point, which allows one to properly position the optic nerve at the origin on the Y-axis. Identifying the optic nerve in each data set allows follow-up images to be aligned reliably.
  3. Mapping the extent of the injection site onto the fundus image
    1. Perform post injection rectangular volume OCT to confirm the success of the injection. Follow the method described in 3.6.
    2. Use the Caliper feature in the software to identify the inflection point at the border of the detached retina from a number of b-scans spanning the entire OCT image. Use the method to open calipers described in 4.1.4.
    3. Record the fundus images with the mapped location of the OCT b-scan and corresponding caliper position on the fundus image, which corresponds to its location.
    4. Compile all of the fundus images into a composite image, including the caliper positions, which results in a precise map of the injection site onto the fundus image (Example in Representative Results section, Figure 5A).

5. Intraocular Injections

NOTE: Details on use of the RIS are further elaborated in a recent study23.

  1. Preparing glass injection needles
    1. Autoclave the capillary tubes with filaments in small batches, using the dry cycle.
    2. Use a pipette puller and set-up a program that will produce a glass tip with the sharpest angle, and a diameter in the range of 2-5 µm.
      NOTE: A sample 5 step program which produced effective needles on our pipette puller is shown in Table 1.
    3. Store the pulled glass needles in a sterile pipette needle jar at room temperature.
  2. Fill the injection needle with the desired solution for injection
    1. Mount the needle into the needle holder, leaving approximately 5/8" protruding beyond the end of the holder.
      NOTE: A distance less than 5/8" will make it difficult reach the injection solution to fill the needle, because the needle holder does not fit readily into the opening of 0.2 mL tubes. Also, having the needle protrude more than 5/8" results in significantly larger oscillation or precession of the tip, making real time imaging difficult since the tip leaves the focal plane or the field of view (FOV), while attempting to puncture the eye.
    2. Prepare the injection solution in a sterile 0.2 mL tube. Add a 1:10 dilution of sterile fluorescein sulfate (10 mg/mL in 1x PBS) to the injection solution to obtain a final concentration of fluorescein at 1 mg/mL.
      NOTE: The exact dye used is specific to the user and can be anything that is non-toxic and visible to the naked eye under white light illumination to help facilitate precise placement of the needle tip at the level of the RPE and subretinal space.
    3. Visualize the needle through the stereo microscope while using the control knobs of the 3-axis micromanipulator to position the needle so that it is aligned with the center of the tube containing the injection solution to be used for filling the needle.
    4. Carefully drive the needle tip into the 0.2 mL tube until the tip of the needle is submerged in the fluid. Draw up the desired volume of injection fluid (e.g. 1 µL) into the needle necessary for a single injection. Maintain the remaining solution in the tube placed in ice.
  3. Preparing the animal for injection
    NOTE:     The RIS microscope is kept clean, and is a non-contact system. The heating plate is covered with a clean adsorbent pad and needles are autoclaved prior to being pulled. After they are pulled by a self-sterilizing heated metal strip, they are kept in a closed sterilized chamber designed to hold pulled glass needles. The needles are only handled using gloved hands, and care is used to prevent touching the tip of the needle while mounting it into the holder. The injected solutions are prepared using sterile technique, and are tested for contamination by streaking a sample of the virus preparations onto LB agar plates and incubating them over night at 37 ˚C.
    1. Weigh the animal (g) to determine the appropriate dose of anesthetic analgesic.
    2. Administer anesthetic (2.5% solution of buffered 2,2,2-tribromoethyl alcohol (Avertin)) via intraperitoneal injection (IP).
    3. Immediately apply anticholinergic drugs (e.g., cyclopentylate) to both eyes to dilate the pupils.
    4. Trim the animal's whiskers and number the animal using ear punch or other method.
    5. Wash the eye and surrounding area with diluted betadine.
      NOTE: Avoid getting any solution around the nose, as this can result in unintentional drowning.
    6. Place the mouse on the heating pad, maintained at 39 ˚C, in the pre-molded modeler's clay mouse holder with the eye to be injected towards the needle.
    7. Make sure the mouse is unresponsive using a pinch test of the hind foot.
  4. Performing subretinal injection
    1. Use a pair of sterile blunt iris forceps to gently induce proptosis of the globe by placing the tips of the forceps at 7 and 10 o'clock positions on the eye lids while pushing open and downward at the same time.
    2. Use the forceps to coax the eyelids under the eye globe to keep it out of the socket during the injection process.
      NOTE: Animals 10 to 14 days old will often hold the eye out of the socket more easily than older animals.
    3. Direct the tip of the needle about 1-1.5 mm below the edge of the corneal limbus and carefully drive it into the eye through the conjunctiva until it creates a scleral depression as the needle penetrates into the tissue of the sclera, and allows manipulation of the eye.
    4. Rotate the eye downward with the micromanipulator to visualize the scleral depression through the dilated pupil with the RIS stereo microscope.
    5. Apply a drop of sterile eye gel or 1x PBS solution and cover with a sterile coverslip.
    6. Focus on the scleral depression created by the tip of the needle (2-5 µm), and drive the needle forward until a sharp peak of the retina forms at the injection site.
    7. Rotate the needle using the holder until the tip bores through the sclera and the fluorescein in the needle is visible under the retina in the immediate vicinity of the RPE cell monolayer.
    8. Drive the tip of the needle so it is tangential to the globe, and then activate the injection pump with the foot pedal switch.
    9. After the desired volume has been delivered (0.5-1 µL) to the subretinal space, withdraw the needle and check if the bleb is stable and that fluid does not leak out of the injection site, which is the first criteria of a successful injection.
      NOTE: Maintaining a proper tip diameter (2-5 µm diameter) is critical to avoid leakage from injection site.
    10. Place the animal on the imaging platform of the OCT instrument. Follow the directions for HR-SD OCT Imaging to record a rectangular volume image.
    11. Confirm that the injected fluid is located in the subretinal space, and save the images for determining the extent of the bleb.
    12. Remove the animal from the holder and apply an ample amount of antibiotic ointment to the injected eyes.
    13. Place the animal onto a heating pad until it completely recovers, then place it back into the original cage where it came from.
      NOTE: Our animals are usually injected prior to weaning, therefore the animals need to be returned to the cage with the mother.
  5. Mapping the extent of the subretinal bleb using OCT instrument software tools.
    1. Obtain a rectangular volume OCT image, using methods described earlier, of the bleb and position it to include a recognizable landmark within the eye such as the ONH.
      NOTE: Recording 90 b-scans works well for mapping the bleb, but could be used depending upon the desired resolution.
    2. Add a single caliper to the figure and place it at the inflection point where the retina detaches from the RPE and choroid.
      NOTE: The software automatically maps a corresponding point to a line onto the fundus image representing the caliper and the b-scan being evaluated.
    3. Capture the screen following placement of the calipers, and compile the images to effectively map the border of the injection bleb onto the fundus image, which precisely maps the injection site. Save the compiled image for reference to help in locating the regions of interest during follow-up imaging.
      NOTE: Alternatively, the caliper data can be saved, complied, and plotted using a similar method for plotting 3D data sets of ONL thickness.
  6. Intravitreal Injection
    1. Place the tip of the needle using a similar surgical approach as sub retinal injection, except that the needle is driven entirely through the sclera at the pars plana about 0.25 mm behind the corneal limbus and into the vitreous.
    2. Following needle placement, apply the injection pulse with the foot pedal.
      NOTE: A rapid diffusion of the fluorescein dye throughout the vitreous of the eyecup is observed, filling the dilated pupil aperture with fluorescence.

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Results

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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 human ...

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Discussion

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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...

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Disclosures

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Commercial Relationships: MCB: None; JMS: None. The retinal imaging system (RIS)23 used in this study is a novel device of substantial use to any group seeking to conduct gene therapy delivery studies in mice, rodents, or small animals. While the authors have no conflicts to declare with respect to this device at this time, the University at Buffalo- SUNY and the Veterans Administration have rights in the intellectual property and may seek to commercialize this instrument in the future.

Acknowledgements

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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 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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
C57BL6 (J)Jackson Laboratories664
N129R-N/AN/A
2HRho 1T/1TN/AN/A
Envisu R-2200 Ocular Coherence Tomography Instrument (OCT)Bioptigen90-R2200-U1-120.  
Retinal Imaging System (RIS)In-houseN/A
Stemi 2000C MicroscopeZeiss000000-1106-133
P-97 Flaming/Brown micropipette pullerSutter Instrument Cop-97
MMN-33 micro manipulator Narishige USAMMN-33
PLI-100  micro injectorHarvard Apparatus64-1736
Micropipette Holder (Rotating)In-houseN/A
Micropipette Storage ReceptacleWorld Precision Instruments Inc.E210
 Borosilicate glass capillary tubes 1.5-1.8 X 100mm, Harvard Apparatus30-0053
2,2,2-TribromoethanolSIGMA AldrichT48402-25G
Tert-amyl AlcoholSIGMA Aldrich240486-100ML
Atropine Sulfate Ophthalmic Solution, USP 1%Akorn Inc.NDC 17478-215-05
GonioviscBioVision LimitedNDC 17238-610-15
Cyclopentolate Hydrochloride Ophthalmic Solution, USP  2%Akorn Inc.NDC 17478-097-10
Gentamicin Sulfate Ointment USP, 0.1%PerigoNDC 45802-046-35
Systane UltraAlcon Laboratories, Inc.9006619-1013
Tetracaine HydrochlorideBausch and LombNDC 24208-920-64
Ophthalmic Solution, USP 0.5%Bausch and LombNDC 24208-920-64
DPBSGibco Life Technologies14190-136
Virus PreparationsViGene /UNCN/A
Gold nanorodsNANOPARTzD12M-850-1.75
Fluorescein Sulfate AK-FLUOR 25%Akorn Inc.NDC 17478-250-20
CoverslipsFisher Scientific12-548-A
ForcepsMilton18-825
Needles 30 guageBeckton Dickenson  W11604
SyringesBeckton Dickenson  309659
Bioptigen software PackageBioptigenN/A
Proparacaine Hydrochloride Ophthalmic Solution, USP 0.5%Akorn Inc.NDC 17478-263-12
Windows ExcelMicrosoftN/A
Adobe IllustratorAdobe 
ScaleMettler
ScissorsWorld Precision Instruments
Ear punchNat’l band
CL 100 Light sourceWelch AllynCL100
Nitrogen GasJackson Welding SupplyN/A
Heated Water bathNeslabRTE-140
Heating plateIn HouseN/A
Heating matCincinnatti Sub Zero273
Clay mouse holderPlast.i.clay American Art Clay Co.N/A
BetadineMedLine NDC53329-938-06
Cotton Tip ApplicatorsAmerican Health ServiceCtag
EtOH 70%Fisher ScientificBP2818-100
Gloves NitrileVWR89038-272
Diagnosys ERG Color Dome instrumentDiagnosys Inc.D125
Contact lensesIn-houseN/A
Diagnosys SoftwareDiagnosys Inc.N/A
Origin 6.1 softwareOriginLab Corp.N/A
Reference electrodesOcuscienceF-Thread Electrode (DTL) 24”

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Subretinal InjectionMouse Eye ImagingOuter Nuclear LayerPhotoreceptor DegenerationAdeno Associated VirusReal Time VisualizationTransscleral ApproachRetinal Imaging System

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