Gonzalez-Fernandez F et al. (2011), Novel strategy for subretinal delivery in Xenopus.

XB-ART-44590

Mol Vis 2011 Mar 23;17:2956-69.

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Novel strategy for subretinal delivery in Xenopus.

Gonzalez-Fernandez F , Dann CA , Garlipp MA .

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The subretinal space, which borders the retinal pigment epithelium (RPE), photoreceptors, and Müller cells, is an ideal location to deliver genetic vectors, morpholino oligos, and nanopharmaceuticals. Unfortunately, materials injected into the space tend to stay localized, and degenerative changes secondary to retinal detachment limit its usefulness. Furthermore, such injection requires penetration of the sclera, RPE/choroid, or the retina itself. Here, we developed a strategy in Xenopus to utilize the continuity of the brain ventricle and optic vesicle lumen during embryogenesis as a means to access the subretinal space. Wild-type and transgenic embryos expressing green fluorescent protein under the rod-opsin promoter were used for optic vesicle and brain ventricle injections. For injection directly into the optic vesicle, embryos were laid on one side in clay troughs. For brain ventricle injections, embryos were placed standing in foxholes cored from agarose dishes. Linear arrays with each embryo positioned dorsal side toward the micromanipulator facilitated high throughput injections. Twenty-five micrometer micropipettes, which were positioned with a micromanipulator or by hand, were used to pressure inject ~1.0 nl of test solution (brilliant blue, India ink, fluorescein isothiocyanate dextran, or 0.04 µm of latex polystyrene microspheres [FluoSpheres®]). FluroSpheres® were particularly useful in confirming successful injections in living embryos. Anesthetized embryos and tadpoles were fixed in 4% paraformaldehyde and cryoprotected for frozen sections, or dehydrated in ethanol and embedded in methacrylate resin compatible with the microspheres. Direct optic vesicle injections resulted in filling of the brain ventricle, contralateral optic vesicle, and central canal. Stages 24 and 25 gave the most consistent results. However, even with experience, the success rate was only ~25%. Targeting the vesicle was even more difficult beyond stage 26 due to the flattening of the lumen. In contrast, brain ventricle injections were easier to perform and had a ~90% success rate. The most consistent results were obtained in targeting the diencephalic ventricle, which is located along the midline, and protrudes anteriorly just under the frontal ectoderm and prosencephalon. An anterior midline approach conveniently accessed the ventricle without disturbing the optic vesicles. Beyond stage 30, optic vesicle filling did not occur, presumably due to closure of the connection between the ventricular system and the optic vesicles. Securing the embryos in an upright position in the agarose foxholes allowed convenient access to the frontal cephalic region. On methacrylate sections, the RPE-neural retina interphase was intact and labeled with the microspheres. As development continued, no distortion or malformation of the orbital structures was detected. In green fluorescent protein (GFP), transgenic embryos allowed to develop to stage 41, retinal FluoSpheres® labeling and photoreceptor GFP expression could be observed through the pupil. On cryosections, it was found that the FluoSpheres® extended from the diencephalon along the embryonic optic nerve to the ventral subretinal area. GFP expression was restricted to rod photoreceptors. The microspheres were restricted to the subretinal region, except focally at the lip of the optic cup, where they were present within the retina; this was presumably due to incomplete formation of the peripheral zonulae adherens. Embryos showed normal anatomic relationships, and formation of eye and lens appeared to take place normally with lamination of the retina into its ganglion cell and the inner and outer nuclear layers. Diencephalic ventricular injection before stage 31 provides an efficient strategy to introduce molecules into the embryonic Xenopus subretinal space with minimal to the developing eye or retina.

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Species referenced: Xenopus laevis

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	Figure 1. The relationship between the optic vesicle and brain ventricles in the developing Xenopus embryo are shown in these diagrams. The cross-sections, which are through the optic vesicles (dashed lines), compare stages 23, 26, and 27 in A, B, and C respectively. These drawings, were prepared based on [24,30], to illustrate that the brain ventricle and optic vesicle lumens are continuous at least at stage 23.
	Figure 2. This diagram illustrates the position of embryos in agarose dishes for brain ventricle injections. Embryos were placed in foxhole arrays punched out of 1% agarose dishes (blue). At least 25 foxholes can be prepared per dish. The holes are made to a depth so that the head protrudes slightly out of the foxhole (see Methods). The embryonic diencephalic ventricle is located along the midline, and protrudes anteriorly just under the frontal ectoderm and prosencephalon (compare with Figure 3). A midline anterior approach conveniently accesses the ventricle without disturbing the optic vesicles (middle embryo). The injected material (pink) readily fills the brain ventricle with optic vesicle, and typically can be appreciated extending into the central canal [30].
	Figure 3. Horizontal section of a stage 26 embryo illustrating the brain ventricle injection method used to introduce materials into the optic vesicle lumen. A: A glass micropipette was introduced into the diencephalic ventricle anteriorly. B: The dashed line shows the orientation of the section in panel A. The drawings were prepared based on [24,30].
	Figure 4. Xenopus subretinal delivery strategy. Animation 1. The attached movie illustrates the diencephalic injection strategy to introduce microspheres into the optic vesicle of Xenopus laevis embryos. Note that the slide bar at the bottom of the quicktime movie can be used to manually control the flow of the movie. If you are unable to view the movie, a representative frame is included.
	Figure 5. Optic vesicle fluorescein isothiocyanateâdextran injection. The injection was performed at stage 24, and photographed here at stage 28. A: Fluorescence shows filling of the optic vesicle (arrow), brain ventricle (black asterisk), and central canal (white asterisk). B: This illustration was prepared to be consistent with stage 28 as in reference [30].
	Figure 6. Cy3 microspheres were injected into the diencephalic ventricle at stage 26. Panels A, B and C, D are dorsal and lateral views, respectively, of the same embryo showing the optic vesicles, which normally appear to bulge laterally from the head (slanted arrows). Under fluorescence, the optic vesicles are filled with FluoSpheresÂ® (panels B, D). The vertical arrow in panels C, D indicates the diencephalic ventricle which also contains the FluoSpheresÂ®. The scale bar in the lower right equals 0.5 mm.
	Figure 7. Texas red microsphere were delivered to retinal pigment epithelium/retina interface. The contralateral optic vesicle was injected with the microspheres at stage 26. A: At stage 28, the embryo was embedded in methacrylate polymer. At this stage, formation of the optic cup is complete and melanin pigment is visible in the retinal pigment epithelium. B: The same section photographed under fluorescence shows the microspheres appearing as red dots (arrows) along the RPE/retina interface.
	Figure 8. FluoSphereÂ® labeling in the tadpole eye. Four days prior, this living, free-swimming tadpole received a Cy3-FluoSpheresÂ® brain ventricle injection at stage 26. A: Macroscopically, there is no apparent distortion or malformation of the orbital structures. B: Under fluorescence, the label is restricted to the brain and retina. In the eye, the fluorescence emanates through the pupil (arrow). Although the right eye was equally labeled, its fluorescence is not visible, as its pupil is not in view.
	Figure 9. Cy3-FluoSpheresÂ® were injectedbefore, and after separation of the optic vesicle lumen from the brain ventricular system. These photographs were taken 10 min after injection into the diencephalic ventricle. A: The older stage 31 (upper) embryo has a more advanced overall body shape, with fuller development of its tail bud (*) compared to the stage 29 embryo beneath it. The arrows show the optic vesicles. B: Under fluorescence, although brain ventricle labeling is seen in both embryos, optic vesicle filling is seen only in the stage 29 embryo (arrows).
	Figure 10. Retina access is dependent on the embryonic stage. The diencephalic ventricles were injected at stage 24 (A, B) or stage 31 (C, D), and the embryos allowed to mature to swimming tadpoles. The eyes of both tadpoles appear to have developed normally. Cy3 fluorescence, which is appreciated through the pupil (arrow), is present only in the stage 24 injected tadpole. Such retinal labeling can be achieved through stage 29/30. For stages 31 and older, brain ventricle injection does not support delivery to the retina.
	Figure 11. Diencephalic ventricular injection was used to deliver Cy3-FluoSpheresÂ® to the retina of transgenic tadpoles expressing green fluorescent protein under the rod-opsin promoter. The brain ventricle was injected through the anterior prosencephalon at embryonic stage 27, and development allowed to continue for 48 h to stage 41. A: In vivo photograph showing the normal external appearance of the eye and pupil (arrow). B: Cy3-FluoSphereÂ® labeling of the retina can be viewed through the pupil. C: Retinal expression of green fluorescent protein (GFP) is apparent through the pupil. D: Cryosection (transverse plane) through the eye at the level of the optic nerve (arrowhead) shows that the Cy3-FluoSpheresÂ® have diffusely labeled the diencephalon (asterisk), and are found extending through the optic nerve to the subretinal region. E: This higher magnification of boxed area in panel D shows GCL, ganglion cell layer; inner nuclear layer, ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigmented epithelium; Arrowheads, rod photoreceptors expressing GFP.

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