Oxybuprocaine hydrochloride

The mouse model of corneal wound injury was generated as described previously²⁸ . Briefly, the day before the wound was made, 1% atropine eye drops (Nitten, Japan) were applied to avoid damage to the iris. After local anesthesia with oxybuprocaine hydrochloride (Santen Pharmaceutical, Japan), a trephine was used to introduce a 2 mm diameter wound in the cornea of the right eye. Epithelial defects were created using a spatula (INAMI Inc.). Each corneal wound was visualized with 1% fluorescein and monitored after surgery every 8 hr until the wound was completely healed. After wounding, levofloxacin eye drops (antibiotics, Santen Pharmaceutical, Japan) were used to prevent corneal infection. The area of the epithelial defect was measured using the ImageJ software. For diclofenac treatment, diclofenac was dissolved into PBS at a concentration of 0.1% (w/v). Diclofenac administration (4 times/day) started 2 days before the wound was made.

As described previously (55 (link)), animals were dark adapted overnight and then anesthetized with ketamine hydrochloride (80 mg/kg) (Daiichi Sankyo Propharma) and xylazine hydrochloride (16 mg/kg) (Bayer Medical). After corneal treatment with 0.4% tropicamide (Nitten) and 0.4% oxybuprocaine hydrochloride (Santen Pharmaceutical), a contact lens electrode was placed on the eye, and reference and ground electrodes were placed in the mouth and on the tail, respectively. ERG was recorded with a PuREC system (Mayo Corporation) on P19.

After assigning an identification number to each animal, the pupils were dilated (0.5% proxymetacaine hydrochloride; Alcon, Bornem, Belgium), and the cornea was anesthetized (4% oxybuprocaine hydrochloride; Santen, Osaka, Japan). The cycloplegic refractive error of both eyes was examined using a streak retinoscope (Keeler, Windsor, UK) in a dark room. Then, A-scan ultrasonography (11 MHz; Optikon HiScan A/B) was performed to measure the lens thickness (LT), axial length (AL), and vitreous chamber depth (VCD). At the end of the myopia induction period, the animals were euthanized using an overdose of sodium pentobarbital, and the eyes were enucleated. Under a surgical microscope (Carl Zeiss, Aalen, Germany), we used a razor blade to collect the tissues on an ice-cold plate by making a cut on the eye perpendicular to the anterior or posterior axis at a location approximately 1 mm posterior to the ora serrata. We then separated the iris and ciliary body of the anterior segment. The retinas were dissected in ice-cold phosphate-buffered saline (PBS) and immersed in an Allprotect Tissue Reagent (Qiagen, Valencia, CA, USA). All the tissue samples were stored at −80 °C prior to RNA extraction.

All ICL-V4c procedures were performed by the same surgeon (XYW) under cycloplegia (2.5% phenylephrine and 1% tropicamide, Alcon, China) and topical anaesthesia (0.4% oxybuprocaine hydrochloride, Santen, Japan). A lid speculum was used to fully expose the ocular surface after the sterilization of the ocular surface and periocular skin (povidone-iodine 5%). Then, a 3 mm incision was created at the temporal or superior corneoscleral limbus. After a viscoelastic surgical agent (1.7% sodium hyaluronate; Bausch & Lomb, China) was injected into the anterior chamber to maintain the anterior chamber depth, an ICL-V4c was inserted into the anterior chamber through the incision with an injector cartridge. An additional viscoelastic agent was then placed on the top of the ICL (between the corneal endothelium and the upper surface of the ICL-V4c), and an ICL positioning instrument was used to sweep the 4 footplates of the ICL beneath the iris. Subsequently, both the anterior chamber and the posterior chamber were irrigated thoroughly using a balanced salt solution. Finally, the overfilled perfusate was released from the incision, and the IOP was estimated with “finger touch” before the end of the procedure.

The OHT mouse model was established as described in our previous study [13 (link), 20 ]. Specifically, the animals were anesthetized by intraperitoneal injection of 1.25% tribromoethanol (20 µl/g, Lab Anim Sci-Tech, China) for dilating pupils and anesthetic eye drops with tropicamide phenylephrine (Santen, Japan) and oxybuprocaine hydrochloride (Santen, Japan). After anterior chamber penetration, we injected 2 µL air and 3 µL microsphere suspension (1 × 10⁵ microspheres/µL, 10 μm diameter, Thermo Fisher Scientific, USA) into the anterior chamber to establish the OHT model. Using a handheld tonometer (TonoLab, Finland), IOP was measured once every six days for 17 days at approximately the same time during at the 9 AM and 11 AM. Six measurements from each eye were recorded and averaged. To explore the protective effects of ADSC-EVs, KM mice were split into the following categories at random (n = 20): Control: sham; OHT + ADSC-EVs: OHT mice treated with intravitreal injection of 3 µg (4.5 × 10⁸ particles) ADSC-EVs in 1.5 µl PBS, and OHT + PBS: OHT mice treated with intravitreal injection of 1.5 µl PBS. ADSC-EVs and PBS were administered intravitreally as described in our previous study by using a 33G Hamilton syringe (Hamilton, USA).

Before surgery, the eyes were instilled with compound tropicamide eye drops (Santen, Japan) 4 times to expand the pupil diameter to greater than 7 mm. Oxybuprocaine hydrochloride (Santen, Japan) eye drops were applied 4 times as topical anaesthesia. A small OVD amount (1.7% sodium hyaluronate, Shandong Bausch and Lomb Freda Company) and balanced salt solution (BSS, Alcon, USA) were injected into the ICL implant container. The injector was properly installed and placed in BSS for later use. An assisted right-side incision was made, through which an appropriate OVD amount was injected into the AC. A 3.0 mm clear corneal main incision was made, typically placed on the steep axis of the corneal curvature, to release corneal astigmatism. The ICL was inserted from the main incision of the AC, and a sufficient OVD amount was injected into the front surface of the ICL to maintain the operating space. After gently tucking the footplate beneath the iris and adjusting the ICL to a central position, the OVD was manually irrigated from the AC using BSS by an injection needle attached to a syringe. Finally, the incisions were made watertight.

Adenoviruses encoding HDAC6 were generated using the pDC315‐Flag vector (GeneChem, Shanghai, China). For adenovirus‐mediated overexpression of HDAC6 in the retina, 8‐week‐old C57BL/6J mice were anaesthetized with 2% isoflurane, and one drop of 0.5% oxybuprocaine hydrochloride (Santen, Osaka, Japan) was applied to the surface of the cornea. The iris was dilated using 0.5% tropicamide phenylephrine (Santen), and mice were intravitreally injected with 1 µL solution containing 4 × 10¹⁰ PFU mL⁻¹ adenoviruses using a 34‐gauge Hamilton needle and syringe (Hamilton Company, Reno, NV) for the following 5 days.
For intravitreal injection of drugs, mouse eyes were treated using the above method. Tubastatin A (sml0044; Sigma‐Aldrich, St Louis, MO) was dissolved in DMSO (0.2 × 10⁻³ m) and diluted 1:10 with 0.9% saline; NQDI‐1 (sml0185; Sigma‐Aldrich) was dissolved in DMSO (1.2 × 10⁻³ m) and diluted 1:10 with 0.9% saline; cytochalasin D (ab143484; Abcam) was dissolved in DMSO (0.1 × 10⁻³ m) and diluted 1:10 with 0.9% saline; prostaglandin E2 (P0409; Sigma‐Aldrich) was dissolved in DMSO (0.5 × 10⁻³ m) and diluted 1:10 with 0.9% saline. Mice were treated by intravitreal injection with 0.5 µL of drugs or the same volume of vehicle every 2 days from P14, when they opened their eyes, until the study endpoint.