Methodology of trans-corneally subretinal injection in mice

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Background Trans-corneally subretinal injection in rodent model is a useful method for genetic therapy, stem cell transplantation and the study on the ophthalmic research. Standarized operation process is critical for the successful treatment. However, there is no literature to report the detailed procedure and the influence of this technique on morphology and function of retina. Objective This sudy was to introduce a method of trans-corneally subretinal injection and evaluate its influence on the morphology and function of retina. Methods Trans-corneallly subretinal injection was performed on the left eyes of 2-month-old SPF C57BL/6J mice after dilation of pupils. A 301/2G disposable needle was used to puncture the cornea within the pupil area near limbus and avoid touching the lens and irises under eye surgery microscope. Then, a 33G blunt needle was used to insert into the vitreous and toward subretinal space via corneal puncture. Normal saline with 0.1% fluorescein sodium of 1 μl was slowly injected into the space, and 2.5% hydroxypropyl methylcellulose was dropped on ocular surface for the observation of the fundus clearly. According to the percentage of the retina filled with subretinally injected solution, the experimental eyes were divided into 80%-100% area group, 50%-70% area group after injection, and the mice in the pseudo-injected group, in which injection procedure stopped just before the solution was pushed in to the subretinal space did not inject any solution after punctured. The right uninjected eyes of the mice served as normal control group. Four eyes were selected for each group. The structural changes were evaluated by optical coherance tomography (OCT) 1 day, 2 days, 3 days and 5 weeks after injection, and retinal function was assessed by the recored of electroretinography (ERG) 5 weeks after injection. The retinal sepcimens were prepared to examin the morphological changes by hematoxylin and esosin staning. The use of care followd the Regulations for the Administration of Affair Concerning Experimental Animals of Zhejiang Province. Results About 70% of the injected eyes showed that retinal blebs filled with injected green fluorescein solution occupied 50% or more retinal area with minimal damages. The focal detachment between neurosensory retinal layer and retinal pigment epithelium (RPE) was exhibited 1 day postinjection, and almost all the retinas retached 2 days after injection. In the fifth week after injection, the amplitudes of ERG b wave were (386.25±37.88), (357.50±41.03), (324.25±53.45) and (410.50±14.88)μV in the sham operation group, 50%-70% area group, 80%-100% area group and normal control group, respectively, showing a significant difference among the 4 groups (F=3.574, P=0.047), and the amplitudes of b wave in the normal control group were higher than those in the 80%-100% area group (all at P<0.05). The detachment between retinal neuroepithelium layer and RPE layer, cell proliferation and transposition in the outer nuclear layer were dispalyed under the light microscope in the sham operation group, 50%-70% area group and 80%-100% area group, and the disordered outer segment of photoreceptors at the injecting area was seen in the 50%-70% area and 80%-100% area groups at five weeks after injection. However, retinal sructure and morphology were normal at the non-injection area. Conclusions Trans-corneally subretinal injection is an effective and safe way for subretinal injection. Key words: Injections/methods; Ophthalmologic surgical procedures/methods; Retina/pathology; Tomography, optical coherence; Electroretinography; Mice, inbred C57BL