Ocular Therapeutic Delivery and Advanced Tissue Retrieval in Adult Rats

Abstract

Therapeutic delivery to the posterior segment of the eye, including the retina and optic nerve, is complicated by the presence of blood-brain and blood-retinal barriers. Small animal models, such as rats, are utilized for studying various ocular pathologies. While therapeutic delivery to the posterior eye is challenging, achieving it is essential for treating ocular disorders, many of which require validation in small animal models for translational relevance. Therefore, two posterior therapeutic delivery techniques are presented: intravitreal injection (IVI) and retrobulbar injection (RBI) for use in adult rats. Additionally, a method for the en bloc removal of the eyes and optic nerves is introduced for various histological and molecular analysis techniques. The dissection protocol enables full observation of the neuro-visual system while minimizing post-mortem injury to retinal and optic nerve tissues. Successful delivery of the therapeutic cyclosporine to the retina and optic nerve was achieved, with detectable concentrations observed twenty-four hours after injection using both IVI and RBI. Furthermore, en bloc retina and nerve samples were successfully extracted for full eye histological tissue analysis, facilitating comprehensive observation of the retina and the wider neuro-visual system.

Figure 1:. Schematic of intravitreal (IV) injection technique.

A 10 μL syringe with a 33 gauge needle, length of 10 mm, and angle of 15 degrees is inserted 2/3 of the way into the eye at the limbus. Created with BioRender.com

Figure 2:. Schematic and representative image of retrobulbar (RB) injection technique.

A) Schematic image of the RB injection. A 0.5 mL insulin syringe with a 28 gauge needle is inserted along the lower orbital rim at angle between 6 and 7 o’clock. Needle is advanced until the back of the ocular socket is felt, and then pulled back slightly before injection. Created with BioRender.com B) Representative image of the RB injection technique using the same needle with black tattoo ink during pilot trials.

Figure 3:. Schematic of the major dissection points during the ocular tissue isolation.

Created with BioRender.com Dotted lines indicate incision points. A) Rat displayed dorsally with atlanto-occipital joint marked. B) Rat skull displayed dorsally with important incision marks noted.

Figure 4:. Flash visual evoked potentials (fVEPs) of RBI saline treated cohorts.

A) Averaged fVEP amplitudes from saline RBI treated right eyes (OD) and control/non-treated left eyes (OS). No significant differences were detected in the ampltidue of fVEP waveforms between groups or between eyes. B) Right eyes (OD) fVEP waveforms of saline RBI treated eyes at baseline and then at seven days after injection (n=2 animal samples).

Figure 5:. Epifluorescent micrographs of the optic nerve head (A,B) and en bloc eye sample (C) of rodents collected following the Ocular Tissue Isolation Dissection.

A-B) Intact optic nerve heads were collected from Sprague Dawley animals and were stained with markers for β-tubulin (green), glial fibrillary acidic protein (GFAP; red), and nuclear marker DAPI (blue) at 20x magnification. C) En bloc eye sample showing the intact globe and full optic nerve.