The crystalline lenses of rabbits were extracted by means of a standard phacoemulsification technique using the White Star phacoemulsification system (Abbott Medical Optics, Santa Ana, CA, USA) [37 (link)]. Surgeries were performed through 2.8-mm clear corneal incisions. To achieve mydriasis, tropicamide 1% (Alcon Laboratories, Geneva, Switzerland) and phenylephrine hydrochloride 2.5% (Alcon Laboratories, Geneva, Switzerland) eye drops were administered approximately 30 min before lens extraction surgery. Corneal incisions were closed with 10/0 nylon sutures, and the rabbits were left aphakic with an intact posterior capsule for at least one week before the experimental cell-injection procedures.
Phenylephrine hydrochloride
Manufactured by Alcon
Sourced in United States
Phenylephrine hydrochloride is a chemical compound used in various laboratory applications. It is a white, crystalline powder that is soluble in water and alcohol. The core function of phenylephrine hydrochloride is to serve as a reagent or standard in analytical procedures, where it can be used to calibrate instruments or verify the performance of analytical methods.
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Lab products found in correlation
Tropicamide, by Alcon (11 mentions)
White star phacoemulsification system, by Abbott (3 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (3 mentions)
Viscoat, by Alcon (3 mentions)
Ketamine hydrochloride, by Zoetis (2 mentions)
Ketamine, by Troy Laboratories (1 mentions)
Proparacaine hydrochloride, by Alcon (1 mentions)
Genteal eye gel, by Alcon (1 mentions)
Stemi 305 stereomicroscope, by Zeiss (1 mentions)
Alcaine, by Alcon (1 mentions)
Xylazine hydrochloride, by Troy Laboratories (1 mentions)
Nanofil microsyringe, by World Precision Instruments (1 mentions)
33 g needle, by World Precision Instruments (1 mentions)
Whitestar signature, by Abbott (1 mentions)
Invivovue oct system, by Bioptigen (1 mentions)
Wild type c57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Xylazine, by Akorn (1 mentions)
Tropicamide 1 solution, by Alcon (1 mentions)
14 protocols using phenylephrine hydrochloride
1
Rabbit Lens Extraction via Phacoemulsification
2
Primate Ocular Preparation for Electrophysiology
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Monkeys were sedated with ketamine (10 mg/kg) (Troy Laboratories, Glendenning, New South Wales, Australia) and xylazine (1 mg/kg) (Lloyd Laboratories, Shenandoah, IA, USA) injected intramuscularly. Pupil dilation was achieved by the combined topical administration of 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon Laboratories, Fort Worth, TX, USA). Before placing the recording lens, proparacaine hydrochloride (0.5%) (Alcon Laboratories, Fort Worth, TX, USA) and methylcellulose (2.5%) (Akorn, Inc., Buffalo Grove, IL, USA) eye drops were applied to assure local anesthesia and corneal surface hydration, respectively.
3
Intravitreal Injection in C57BL/6J Mice
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Ten- to twelve-week-old C57BL/6J mice were anesthetized with a ketamine/xylazine cocktail (120 mg/kg, 16 mg/kg, respectively) followed by pupil dilation using tropicamide (1% [w/v]) and phenylephrine hydrochloride (2.5% [w/v]), both from Alcon, Fort Worth, Texas. Alcaine (0.5% [w/v], Alcon) was used to locally anesthetize the eye followed by application of GenTeal eye gel (severe dry eye formula, Alcon) to prevent corneal drying. Intravitreal injections were guided by the use of a Stemi 305 stereo microscope (Zeiss, Oberkochen, Germany). Briefly, the right eye was slightly proptosed by periocular pressure and a 33G ½” needle with a 10 to 12° bevel fitted to a Hamilton micro syringe (Hamilton, Reno, NV, USA) was inserted just below the limbus at a ∼45° angle. Two microliters of rAAV (7.6 × 109 viral genomes) was slowly injected into the vitreous over the course of ∼1 minute. For the left eye, the same injection procedure was followed, but using vehicle (Hanks' Balanced Salt Solution with 0.14% Tween [HBSS-T]). Postinjection, a drop of GenTeal and a small amount of AK-POLY-BAC antibiotic ointment (Akorn, Lake Forest, IL, USA) were applied to the eye. Forty-eight hours after injection, mice were macroscopically inspected and those with any apparent ocular damage (e.g., cloudy eye) were not used for subsequent experiments.
4
Genetically Modified Mice for Inflammasome Studies
5
Intravitreal Injection Procedure in Mice
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The procedure for IVT injection has been described previously [29 (link),75 (link),76 (link)]. Briefly, prior to injection animals were anesthetized with 150 mg/kg body weight (BW) ketamine hydrochloride (Parnell Laboratories, New South Wales, Australia) and 10 mg/kg BW xylazine hydrochloride (Troy Laboratories, New South Wales, Australia) and their pupils were dilated with a topical application of 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon Laboratories Inc., Fort Worth, TX, USA). For the injection, a 1 µL aliquot of solution containing each chemical was injected into the vitreous body of both eyes of each mouse using a Nanofil microsyringe with a 33G needle (World Precision Instruments Inc., Sarasota, FL, USA), under an ocular surgery microscope (Leica Microsystems GmbH, Wetzlar, Germany). A bilateral approach was taken to minimize the number of animals used in this study. All procedures were performed carefully to avoid injuring the lens or retina. Four or seven days following injections, the eyes were collected and subjected to further analysis as described below. Eyes with bleeding or inflammation caused by technical errors were excluded from analysis.
6
Rabbit Lens Extraction and Preparation
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The crystalline lenses of rabbits were extracted using a standard phacoemulsification technique as previously described17 (link). Mydriasis was achieved by the administration of tropicamide 1% (Alcon Laboratories, Texas, USA) and phenylephrine hydrochloride 2.5% (Alcon Laboratories) eye drops approximately 30 minutes before surgery. A clear corneal incision was made with a 2.8 mm disposable keratome. A 5.0 mm diameter continuous curvilinear capsulotomy of the anterior capsule was created under viscoelastic material (Viscoat; Alcon Laboratories) instilled into the anterior chamber. Hydro-dissection was performed using a 27-gauge cannula. The lens was then aspirated and removed with a standard phacoemulsification procedure using the White Star phacoemulsification system (Abbott Medical Optics, California, USA). Subsequently, the corneal incision was sutured with 10/0 nylon suture and the rabbits were left aphakic with an intact posterior capsule for at least one week before the CE-CI procedures. Cataract extraction surgery of the rabbits were performed by either HSO or FMW.
7
Anesthesia and Pupil Dilation in Mice
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Both male and female wild-type (WT) C57BL/6J mice between 6 and 10 weeks of age purchased from The Jackson Laboratory (Bar Harbor, ME, USA) were used in this study. For all procedures, anesthesia was achieved by intraperitoneal injection of 100 mg/kg ketamine hydrochloride (Fort Dodge Animal Health, Overland Park, KS, USA) and 10 mg/kg xylazine (Anased, Akorn, Decatur, IL, USA), and pupils were dilated with topical 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon, Fort Worth, TX, USA). All animal experiments were approved by the University of Virginia Institutional Animal Care and Use Committee and was performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.
8
Cataract Extraction Surgery in Rabbits
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For cataract extraction surgery of the rabbits performed by either YCL or MB, tropicamide (1%; Alcon Laboratories, Fort Worth, TX, United States) and phenylephrine hydrochloride (2.5%; Alcon Laboratories) eye drops were administered 30 minutes before the surgery to achieve mydriasis. A clear corneal incision was made with a 2.8 mm disposable keratome. After viscoelastic material containing sodium chondroitin sulphate and sodium hyaluronate (Viscoat; Alcon Laboratories) was instilled into the anterior chamber, a 5.0 mm diameter continuous curvilinear capsulotomy of the anterior capsule was created. Hydro-dissection was performed using a 27-gauge cannula. The lens was then aspirated and removed with a standard phacoemulsification procedure using the White Star Signature® phacoemulsification system (Abbott Medical Optics, Santa Ana, CA, USA). Subsequently, the corneal incision was sutured with 10/0 nylon suture and the rabbits were left aphakic with an intact posterior capsule for at least one week before TE-EK surgery.
9
Ocular Imaging and Retinal Analysis
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Eyes were dilated with 1% atropine, followed by 2.5% phenylephrine hydrochloride (Alcon, Fort Worth, TX), and then mice were anesthetized with ketamine (72 mg/kg)/xylazine (4 mg/kg). One drop of 2.5% hydroxypropyl methylcellulose (Gonak; Akorn, Lake Forest, IL) was applied to each eye before examination. Spectral domain optical coherence tomography (SD-OCT) was performed using the InVivoVue OCT system (Bioptigen, Inc., Durham, NC). Three lateral images (nasal, central, and temporal) were collected, starting 0.2 mm above the meridian crossing through the center of the optic nerve (ON), at the ON meridian, and 0.2 mm below the ON meridian. A corresponding box centered on the ON with eight measurement points separated by 0.2 mm from each other was created. Corresponding neural retinas from different treatments were compared at the same location (0.2 mm temporal to the ON) from the vitreous face of the ganglion cell layer to the apical face of the RPE across the retina.
10
Intravitreal Injection of Alu RNA in Mice
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Wild-type C57BL/6J mice between 6 and 8 weeks of age (The Jackson Laboratory, Bar Harbor, ME, USA) were used in this study. The mice were anesthetized by 100 mg/kg ketamine hydrochloride (Fort Dodge Animal Health, Overland Park, KS, USA) and 10 mg/kg xylazine (Akorn, Lake Forrest, IL, USA). Animals were placed on a heating pad at 37°C, and pupils were dilated using topical 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon, Fort Worth, TX, USA). All animal experiments were approved by the University of Virginia Institutional Animal Care and Use Committee and performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. For this study, the surgeons performed SRI of 1× phosphate buffered saline (PBS), an inert substance, or Alu RNA, which induces RPE degeneration.14 (link)–25 (link) Under a biosafety cabinet, we diluted 10× PBS ultrapure-grade (VWR, Radnor, PA, USA) to 1× PBS; 300 ng Alu RNA was prepared as previously described and injected.14 (link),20 (link)
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