Tobramycin dexamethasone eye ointment

The scAAV5-smCBA-hRPE65 vector as used in previous studies was used to deliver RPE65 gene in rd12 mice, with the same method of subretinal injections at age P14 [18 (link), 21 (link), 22 (link)]. Animals were prepared with pupil dilation and general anesthesia. A 30.5-gauge disposable needle was used to make a small incision in the cornea within the pupil area. Then a 33-gauge, unbeveled, blunt needle mounted on a 5 μL syringe (Hamilton Co., Reno, NV) was introduced through the corneal incision to reach the subretinal space in the inferior central region, avoiding touching the lens and penetrating the neuroretina. One microliter of vector suspension (1 × 10¹³ genome containing particles/mL) containing 1% fluorescein was injected slowly in the subretinal space in the right eye of rd12 mice. The injected retinal area was visualized by fluorescein positive subretinal blebs demarking the retinal detachment and more than 95% retinal detachment indicates successful injection. After injection, 1% atropine eye drops and 0.3% tobramycin-dexamethasone eye ointment (Alcon Laboratories Inc., Fort Worth, TX) were given 3 times a day for 3 days. Animals with any complications, including iris-cornea adhesion, iris or retinal hemorrhage, and lens injury, were excluded from the experiment.

The assays include chloroform (TCM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and PHBV, which were purchased from Sigma (USA). Rosiglitazone was obtained from BioVision (USA). Ammonium formate came from Fisher scientific (USA). Acetonitrile was obtained from Merck Millipore (Germany). Filed emission scanning electron microscope SU8010 was obtained from Hitachi (Tokyo, Japan). LC-20A high-performance liquid chromatography (HPLC) were obtained from Shimadzu equipped with an autosampler (Model SIL-20A), a column temperature controller compartment (CTO-10AS) and ultraviolet detector (SPD-M20A). Diamonsil C18 column (5 μ 250 × 4.6 mm) were purchased from Dikma Technologies Inc., China. PH meter came from Denver Instrument, USA. TonoVet were obtain from Icare, Finland. In the vivo experiments, the assays include pentobarbital sodium (30 mg/kg) (Solarbio, Beijing, China), Benoxil (Santen, Japan), Tobramycin Dexamethasone Eye Ointment (Alcon, USA), 0.5% Levofloxacin Eye Drops (Santen, Japan), 4% Paraformaldehyde (Solarbio, Beijing, China), suture line (10-0 nylon; Alcon; USA), 8-0 Vicryl suture (Ethicon; USA), Normal Saline (Second Xiangya Hospital, Changsha, China), Mitomycin C (Sigma, USA), anti-Collagen I antibody (Abcam, UK), anti-α-SMA antibody (Abcam, UK), and anti-connective tissue growth factor (CTGF) antibody (Abcam, UK).

C57BL/6 mice (8 weeks old; Slaccas, Changsha, China) were fed with standard laboratory food and water in a comfortable environment with a 12 h light–dark cycle. All the experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University (Changsha, China). All mice were divided into Three groups: Sham (only acupuncture without injection), NMDA (20 mM), and MT (20 mM NMDA + 400 mM MT). All the mice were anesthetized with pentobarbital (1%, 80 mg/kg, intraperitoneal injection; Beijing Sanshu, China) and then operated on under a stereomicroscope. Oxybuprocaine hydrochloride (Santen Pharmaceuticals, Tokyo, Japan) was used to induce ocular surface anesthesia, and tropicamide phenylephrine (Santen Pharmaceuticals) was used to dilate the pupils. A 30 G needle was inserted into the vitreous cavity along the limbus and injected at a volume of 1 µL per eye. Tobramycin dexamethasone eye ointment (Alcon Inc, Geneva, Switzerland) was used to prevent infection after injection. The mice were euthanized 5 d after the injection, and their eyeballs were removed with tweezers for the follow-up research.