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.
