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25 protocols using proparacaine hydrochloride

1

Dark-Adapted Electroretinography in Mice

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P24 or adult 6 week old mice were dark-adapted overnight and then anesthetized using ketamine (40mg/kg) and xylazine (8mg/kg). Pupils were dilated using 1% tropicamide (Bausch and Lomb, Tampa, FL, USA), 2.5% phenylephrine hydrochloride (Akorn Inc., Lake Forest, IL, USA), and 1% cyclopentolate (Bausch and Lomb). Eyes were anesthetized with 0.5% proparacaine hydrochloride (Akorn, Inc.). Mice were placed on a temperature regulated heating pad and ERGs were recorded using an Espion E3 ColorDome Full field Ganzfeld (Diagnosys, LLC, Lowell, MA, USA) as described previously (Ng et al., 2010 (link)). Details about rod (scotopic) and wavelength specific photopic responses are mentioned in supplemental experimental procedures.
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2

Laser-induced Glaucoma Model in Rodents

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Animals were anesthetized with a mixture of ketamine/xylazine (100mg/kg and 10mg/kg, respectively, intraperitoneal) on a veterinary heating pad. A drop of topical anesthetic (proparacaine hydrochloride, 0.5%, Akorn, Lake Forest, IL) was applied to the cornea of a randomly selected eye. The laser procedure was performed as we recently published [15 (link)]. Briefly, animals were given monocular laser photocoagulation (532 nm, 300 mW x 0.5 seconds per spot, OcuLight TX, IRIDEX Corporation, Mountain View, CA) of the episcleral veins (70 ~ 80 spots) combined with 270° limbal vessels (sparing nasal 90°, 110 ~ 120 spots). After treatment, antibiotic ointment (Ak-Poly-Bac, Akorn, Lake Forest, IL) was added to the treated eye. The fellow untreated eye served as a within animal control.
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3

Optic Nerve Crush Injury Model

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ONC was performed as previously described [52 (link), 123 (link)]. Briefly, mice were anaesthetized via intraperitoneal injection of ketamine (120 mg/kg) and xylazine (11.3 mg/kg). One drop of 0.5% proparacaine hydrochloride (Akorn, Lake Forest, IL) was used to numb the experimental eye. A lateral canthotomy exposed the optic nerve and was followed by an incision in the conjunctiva at the limbal junction. The optic nerve was clamped for 3 s using self-closing N7 forceps (Roboz Surgical Instrument Co, Gaithersburgs, MD). After surgery, triple antibiotic ointment was used to cover the eye, and a subcutaneous injection of buprenorphine (0.2 mg/kg) was delivered to mitigate pain. The right eye was not surgerized and evaluated as the contralateral condition.
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4

Ischemia/Reperfusion Injury in Mice

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All procedures with animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional animal care and use committee (Animal Welfare Assurance no. A3307–01). All surgeries were performed under anesthesia, and all efforts were made to minimize suffering. We used male WT and A2-deficient (A2−/−) mice on C57BL6J background. These mice were subjected to I/R injury in the right eye. I/R was induced as previously described.11 (link) Briefly, mice (10–12 weeks old) were anesthetized with (73 mg/kg ketamine hydrochloride and 7.3 mg/kg xylaxine hydrochloride, i.p.), 1% tropicamide (Akorn, Lake Forest, IL, USA) was used to dilate the pupil, and topical anesthesia (0.5% proparacaine hydrochloride; Akorn) was applied to the cornea. A 30-gauge needle cannulated in the anterior chamber of the right eye was used to infuse sterile saline. The IOP was raised to 110 mm Hg by elevating the saline reservoir and this pressure was held for 40 min. Ischemia was confirmed by observation of whitening of the anterior segment of the globe and blanching of episcleral veins.55 (link) The left eye was used as control. The mice were killed at various times after I/R as determined in preliminary studies and the existing literature as follows:
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5

Paraquat-Induced Oxidative Stress Model

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Wild type mice (male and female, strain B6;129SF2/J, Jackson Laboratory, Bar Harbor, ME) were used. Oxidative stress injury was induced in the retina at age 8 weeks by subretinal injection of paraquat, which has been used by many groups to simulate the elevated oxidative stress that is observed in retinal degenerations. The mice were first anesthetized by intraperitoneal injection of a xylazine-ketamine mixture and the eyes were locally anesthetized with eye drops containing 0.5% proparacaine hydrochloride (Akorn, Lake Forest, IL). Mice were then subretinally injected with either 2 μl of paraquat (PQ, 1mM) or saline control, as described [13 (link)]. Briefly, an incision was made in the conjunctiva and the sclera and injections were performed using a 1.5 cm 33-gauge Hamilton needle (Hamilton Company, Reno, NV) between the RPE and neural retina. The left eye of each mouse was injected, and all injections were localized in the same area of the eye. All mice received the same volume of injection fluid. A successful injection was indicated by a transient retinal detachment that spontaneously resolved, which was confirmed by OCT analysis at 1 week post-injection. We excluded from the study any mice with unresolved retinal detachments, bleeding or infection.
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6

Reagent Preparation for In Vivo Experiments

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All reagents were purchased and used as received unless otherwise indicated. MCC950 sodium salt was acquired from Selleckchem.com (Houston, TX; Catalog No. S7809) and formulated to 4.4 mg/mL in a vehicle consisting of 10% dimethyl sulfoxide (DMSO, Sigma, Burlington, MA; Catalog No. D8418) and 90% Dulbecco’s phosphate buffered saline (DPBS, Gibco, Waltham, MA; Catalog No. 14190–144). Ketamine (NDC 0409–2051-15) was obtained from Hospira, Inc. (Lake Forest, IL). Xylazine (NDC 593990110–20), proparacaine hydrochloride (NDC 17478–263-12), and fluorescein (NDC 17478–253-10) were acquired from Akorn, Inc. (Lake Forest, IL). Tropicamide (NDC 61314–354-01) was purchased from Sandoz (Basel, Switzerland) and phenylephrine (NDC 42707–102-15) was obtained from Paragon BioTeck, Inc. (Portland, OR). GenTeal Severe gel (0065–8064-01) was purchased from Alcon laboratories, Inc (Fort Worth, Texas).
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7

Intravitreal Voriconazole Thermogel Injection

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The liquid thermogel was vortexed, and then 0.3 mL of the 1.7% (5 mg total) voriconazole-thermogel were drawn into a tuberculin syringe and stored on ice to maintain the liquid state. The suspension was mixed by rocking in the syringe just prior to injection. The injection was performed using a 30 gauge, ½ inch needle, and in all cases a well-defined gel deposit was observed immediately following the injection in the SCo space.
For the injection, the horses were placed in the stocks and sedated with detomidine hydrochloride (0.01 mg/kg, IV) (Dormosedan®, Zoetis Kalamazoo, MI). Auriculo-palpebral and supraorbital nerve blocks were performed by subcutaneous injection of 1 mL of 2% lidocaine hydrochloride (Hospira, Inc., Lake Forest, IL). Local anesthesia using proparacaine hydrochloride 0.5% (Akorn, Lake Forest, IL) was applied topically to the injection site in the dorsal bulbar conjunctiva.
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8

Retinal Excitotoxicity Induction in Mice

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Retinal excitotoxicity was induced according to the method established in our laboratory [26 (link)]. Mice were anesthetized with 73 mg/kg ketamine hydrochloride and 7.3 mg/kg xylazine hydrochloride intraperitoneally. Pupils were dilated with 1% tropicamide (Akorn, Lake Forest, IL, USA). One drop of proparacaine hydrochloride (Akorn) was applied to the cornea as topical anesthesia. NMDA (N-Methyl-D-Aspartate, (Sigma, St. Louis, MO, USA), 20 n moles/eye, 1 µL, dissolved in saline) was injected intravitreally into the right eye using a beveled 35 G needle (NF35BV-2, World Precision Instruments, Sarasota, FL, USA) connected with an SGE Syringe (World Precision Instruments, Sarasota, FL, USA). The needle was moved out slowly after maintaining for 30 s. Antibiotic ointment was applied to prevent infection. NMLA (N-Methyl-L-Aspartate, 20 n moles/left eye) was used as control.
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9

Anesthetic Protocol for Rodent Studies

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Atropine-sulfate (1%, Bausch & Lomb Inc., Tampa, FL) was applied bilaterally to corneal surfaces. Nitroglycerin (5 mg/mL, American Regent, Inc., Shirley, NY) was injected intraperitoneal at 10 mg/kg. Carprofen (50 mg/mL, Pfizer, Inc.) was injected subcutaneously at 5 mg/kg. Inhalant isoflurane (Phoenix), topical proparacaine hydrochloride (0.5%, Akorn, Inc.), ketamine (100 mg/mL, Phoenix) at 120 mg/kg, and xylazine (20 mg/mL, Lloyd Laboratories) at 10 mg/kg were used in anesthesia.
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10

Anesthetized Schirmer Tear Test Protocol

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Tear production was measured using the anesthetized Schirmer Tear Test (STT, Eagle Vision, Memphis, TN, USA). To minimize reflex tearing, one drop of 0.5% proparacaine hydrochloride (Akorn, Lake Forest, IL, USA) was placed onto the corneal surface and excess fluid was absorbed at the medial canthus using eye spear sponges (Fine Science Tools, Foster City, CA, USA). After 5 minutes, the notched strip was inserted into the lower lateral conjunctival fornix, maintaining contact with the lateral cornea. The wetted length (mm) of the strip indicated by blue dye appearance was read after 5 minutes.
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