PMC:2669177 / 33305-35305
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/2669177","sourcedb":"PMC","sourceid":"2669177","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2669177","text":"Subretinal injections\nMice (rds +/− pups at P5) were anesthetized by incubation on ice for 2–2.5 minutes. The eyelid of the right eye was cut, the cornea was exposed, and a puncture in the cornea was made with a sterile 30-gauge needle. A 35-gauge blunt-end needle attached to a 10 µl Nanofil® syringe (World Precision Instruments, Sarasota FL) was inserted into the puncture under an operating microscope (Carl Zeiss Surgical, Inc., NY). A volume (0.3 µl) of solution containing fluorescein dye and either nanoparticles, saline (vehicle), or naked DNA was delivered into the subretinal space, usually in the superior temporal quadrant. Since the retina is not fully developed at this age, some injected material is likely released into the vitreous, although the site of injection is subretinal. After injection, the needle was left in place for 3–5 seconds to allow full treatment delivery before being withdrawn gently. Successful delivery of material was confirmed by observation of subretinal yellow-green fluorescence at the time of injection. The cut eyelid was returned to its original position and the surface of the eye was gently blotted with a Kimwipe. Animals were warmed on a temperature-controlled (37°C) bed until fully awake. All nanoparticles and uncompacted plasmid DNA (naked DNA) were used at the same concentration (3.06 µg/µl), selected based on data from our previous study [13]. Because the compaction process relies on the presence of DNA, there is no “empty” nanoparticle, so controls were limited to saline and naked DNA carrying the same therapeutic vector as the nanoparticles. If material delivery could not be confirmed, or if microphthalmia or signs of intraocular infection were observed, the injection was considered unsuccessful and the animal was removed from the study (121/432∼28%). Mice were maintained in the breeding colony under cyclic light (14-hour light/10-hour dark) conditions; cage illumination was approximately 7 foot-candles during the light cycle.","divisions":[{"label":"title","span":{"begin":0,"end":21}}],"tracks":[{"project":"2_test","denotations":[{"id":"19390689-17183666-97510252","span":{"begin":1399,"end":1401},"obj":"17183666"},{"id":"19390689-17183666-97510252","span":{"begin":1399,"end":1401},"obj":"17183666"},{"id":"19390689-17183666-97510252","span":{"begin":1399,"end":1401},"obj":"17183666"},{"id":"19390689-17183666-97510252","span":{"begin":1399,"end":1401},"obj":"17183666"}],"attributes":[{"subj":"19390689-17183666-97510252","pred":"source","obj":"2_test"},{"subj":"19390689-17183666-97510252","pred":"source","obj":"2_test"},{"subj":"19390689-17183666-97510252","pred":"source","obj":"2_test"},{"subj":"19390689-17183666-97510252","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93af","default":true}]}]}}