All the measurements using the DOCT flowmeter were performed by KT. After the dilation of the pupils using a 0.5% tropicamide eye drop (Santen Pharmaceutical Co., Osaka, Japan), the subjects rested for at least 5 min in a dimly lit room at a temperature of 25 °C and their blood pressure was measured in the left arm. All subjects underwent comprehensive eye examinations, such as BCVA: logMAR (a standard logarithmic visual acuity chart was used), intraocular pressure (IOP), slit-lamp examination, and optical coherence tomography (Triton, Topcon Corp., Tokyo, Japan) to measure the central macular thickness. The mean ocular perfusion pressure was calculated at 2/3 mean blood pressure (MBP)–IOP. At least 24 h before the measurements, the subjects were instructed to refrain from intaking caffeine-containing drinks, such as coffee. RBF was measured using a DOCT flowmeter on three veins, i.e., 1. an occluded vein in BRVO eyes; 2. a non-occluded vein on the opposite hemisphere of the occluded vein in the BRVO eye; and 3. a vein in an equivalent area (supero- or infero-temporal) in the fellow eyes. The RBF was measured at veins at a half disc diameter away from the optic disc.
Tropicamide eye drop
Manufactured by Santen
Sourced in Japan
Tropicamide eye drops are a pharmaceutical product used to dilate the pupil of the eye. The active ingredient, tropicamide, is a short-acting mydriatic and cycloplegic agent that temporarily paralyzes the muscles responsible for pupil contraction and eye focusing.
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Lab products found in correlation
Oxybuprocaine hydrochloride eye drop, by Santen (3 mentions)
Phenylephrine hydrochloride, by Santen (3 mentions)
Tobradex, by Alcon (2 mentions)
Envisu r4310, by Leica camera (2 mentions)
Butorphanol tartrate, by Meiji Seika Pharma (2 mentions)
Medetomidine, by Sandoz (2 mentions)
Visucam 200, by Zeiss (2 mentions)
Triton, by Topcon (1 mentions)
Pentobarbital sodium, by Merck Group (1 mentions)
Xylazine hydrochloride, by Bayer (1 mentions)
Novus spectra, by Lumenis (1 mentions)
Phenylephrine, by Santen (1 mentions)
Chloral hydrate, by Merck Group (1 mentions)
Triformol, by Merck Group (1 mentions)
Tuj 1 antibody, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Proparacaine hydrochloride eye drops, by Alcon (1 mentions)
3d oct 2000, by Topcon (1 mentions)
Ofloxacin ointment, by Santen (1 mentions)
24 protocols using tropicamide eye drop
1
Retinal Blood Flow Measurement in BRVO
2
Retinal Venous Flow Measurement Protocol
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An examiner (TT) performed all measurements. After the pupils were dilated with a 0.5% tropicamide eye drop (Santen Pharmaceutical Co., Osaka, Japan), the subjects were required to rest for at least 5 minutes while sitting in a quiet, dimly lit room at a temperature of 25˚C, after which their BP was measured in the left arm. The DOCT flowmeter was assessed after the BP and IOP measurements. The subjects abstained from coffee for at least 24 hours before the measurements. Considering the measuring limit of vessel diameter using DOCT flowmeter (>50μm), the relatively large veins (diameter of 80 μm or more) chosen for measurement had relatively straight segments one disc diameter away from the optic disc. When there were two or more veins in a sector, the biggest one was chosen for the measurement because smaller veins were more likely to give unsuccessful measurements because veins get smaller after PRP. We measured the D, V, and RBF of the inferotemporal (IT), superotemporal (ST), inferonasal (IN), and superonasal (SN) veins (Fig 2 ), and we defined the SRBF as the sum of all measured veins (IT + ST + IN + SN), and vessel D as the automatically calculated flow image of the horizontal lumen. The waveforms of the veins in the case were shown in Fig 2 .
3
Polystyrene Microspheres Induced Ocular Hypertension
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In this study, we performed a procedure where small volumes of polystyrene microspheres were injected into the anterior chamber to impede aqueous outflow and elevate IOP and 69 mice were used for this procedure. The baseline IOP of each eye was measured a day before the operation. Firstly, the animals were anesthetized with 5% chloral hydrate, the pupils were enlarged with 1% tropicamide eye drops (Santen, Japan) and TobraDex (Alcon, USA) anesthetic drops were added to each eye. A 33-gauge needle was inserted into the anterior chamber through the corneal center and 2 µm polystyrene of the microsphere’s solution was injected. In order to avoid contact between the needle and lens, the needle was inserted at an angle of 45° making sure that the vitreous was filled appropriately. Then, 2 µL of air was injected before pulling the needle to seal the small puncture. On day 3, 2 µL miRNA/NPs-BRZ and 2 µL PBS was injected intravitreally. The control group was only punctured in vitreous region without injection. The baseline IOP was recorded before the injection of polystyrene microspheres at the same time each day using the ophthalmotonometer (Reichert, Depew, NY 14043). The normal IOP of each eye was expressed as an average of values measured by 10 ophthalmotonometers (Suman et al., 2014 (link)).
4
Retinal Safety Evaluation of ART
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Mice were anesthetized with a solution containing ketamine (120 mg/kg), xylazine (20 mg/kg), and saline solution (1:1:8) through intraperitoneal injection. Oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Co. Ltd., Osaka, Japan), tropicamide eye drops (Santen), and ofloxacin ophthalmic ointment (Shenyang Xingqi Pharmaceutical Co., Ltd., Shenyang, China) were applied for topical anesthesia, pupil dilation, and infection prevention, respectively.
For the retinal safety study, 30 mice were divided into 6 groups randomly, including one control group and five groups receiving ART of different concentrations. ART was diluted with sterile saline with final concentrations at 25, 50, 100 μM, and 1 mM. The micro-injector was inserted into the vitreous through the incision made by a 30-gauge needle 0.5 mm posterior to the limbus. Serially diluted ART solutions or sterile saline of 1 μL were then injected into the mouse eye under a surgical microscope. Ocular changes including intraocular inflammation, retinal hemorrhage and cataract were observed through daily ophthalmic examination after the injection.
For the retinal safety study, 30 mice were divided into 6 groups randomly, including one control group and five groups receiving ART of different concentrations. ART was diluted with sterile saline with final concentrations at 25, 50, 100 μM, and 1 mM. The micro-injector was inserted into the vitreous through the incision made by a 30-gauge needle 0.5 mm posterior to the limbus. Serially diluted ART solutions or sterile saline of 1 μL were then injected into the mouse eye under a surgical microscope. Ocular changes including intraocular inflammation, retinal hemorrhage and cataract were observed through daily ophthalmic examination after the injection.
5
Comprehensive Ocular Biometrics of Mice
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AL was measured from the anterior corneal surface to the retinal pigment epithelium using a spectral domain optical coherence tomography (SD-OCT) system (Envisu R4310, Leica, Germany). The refraction was detected using an eccentric infrared photo refractor (Steinbeis Transfer Center, Germany) at the vertical pupil meridian. The data were automatically recorded by the program when the parameter values were stable. Choroidal thickness (ChT) was measured using SD-OCT, and the posterior surface of the choroid was quantified using Image J (Ver 1.53, NIH). ChT was determined using the formula: area divided by circumference. Intraocular pressure (IOP) was measured using a tonometer (Tono Lab, Icare Finland Oy, Vantaa, Finland) calibrated for mice under anaesthesia. Parts images of ocular components measurement were shown in Additional file 5 : Fig. S5.
Mice were anesthetized with 0.75 mg/kg medetomidine (Sandoz K.K., Tokyo, Japan), 4 mg/kg midazolam (Domitor®, Orion Corporation, Espoo, Finland), and 5 mg/kg butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) dissolved in normal saline. Mydriasis was induced by 0.5% tropicamide eye drops (Santen, Osaka, Japan), and mice were placed in a cylindrical holder for measurement.
Mice were anesthetized with 0.75 mg/kg medetomidine (Sandoz K.K., Tokyo, Japan), 4 mg/kg midazolam (Domitor®, Orion Corporation, Espoo, Finland), and 5 mg/kg butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) dissolved in normal saline. Mydriasis was induced by 0.5% tropicamide eye drops (Santen, Osaka, Japan), and mice were placed in a cylindrical holder for measurement.
6
Comprehensive Ocular Biometrics of Mice
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AL was measured from the anterior corneal surface to the retinal pigment epithelium using a spectral domain optical coherence tomography (SD-OCT) system (Envisu R4310, Leica, Germany). The refraction was detected using an eccentric infrared photo refractor (Steinbeis Transfer Center, Germany) at the vertical pupil meridian. The data were automatically recorded by the program when the parameter values were stable. Choroidal thickness (ChT) was measured using SD-OCT, and the posterior surface of the choroid was quantified using Image J (Ver 1.53, NIH). ChT was determined using the formula: area divided by circumference. Intraocular pressure (IOP) was measured using a tonometer (Tono Lab, Icare Finland Oy, Vantaa, Finland) calibrated for mice under anaesthesia. Parts images of ocular components measurement were shown in Additional file 5 : Fig. S5.
Mice were anesthetized with 0.75 mg/kg medetomidine (Sandoz K.K., Tokyo, Japan), 4 mg/kg midazolam (Domitor®, Orion Corporation, Espoo, Finland), and 5 mg/kg butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) dissolved in normal saline. Mydriasis was induced by 0.5% tropicamide eye drops (Santen, Osaka, Japan), and mice were placed in a cylindrical holder for measurement.
Mice were anesthetized with 0.75 mg/kg medetomidine (Sandoz K.K., Tokyo, Japan), 4 mg/kg midazolam (Domitor®, Orion Corporation, Espoo, Finland), and 5 mg/kg butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) dissolved in normal saline. Mydriasis was induced by 0.5% tropicamide eye drops (Santen, Osaka, Japan), and mice were placed in a cylindrical holder for measurement.
7
Elevated Intraocular Pressure Mouse Model
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An RIR model was established as previously described (Hartsock et al., 2016). Briefly, mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg; Merck, Kenilworth, NJ, USA, Cat# P-010-1ML). Procaine eyedrops (4 mg/mL; Santen Pharmaceutical, Shanghai, China) and tropicamide eyedrops (5 mg/mL; Santen Pharmaceutical) were dropped onto the left eye of each mouse for local anesthesia and pupil dilation, respectively. Thereafter, we cannulated the anterior chamber with a 30-gauge needle connected to a 1.2-meter tall saline bottle for 1 hour as shown in Figure 1A Additional Figure 1 Figures 2 3
8
Intravitreal Injection of NMDA and Inhibitors
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Intravitreal injections of NMDA (Sigma-Aldrich Corp.) were performed in the same fashion as previously described. 36 Briefly, the rats were anesthetized with an intraperitoneal injection of a 1:1 mixture of xylazine hydrochloride (4 mg/kg; Bayer HealthCare, Leverkusen, Germany) and ketamine hydro-chloride (10 mg/kg; Daiichi Sankyo Propharma Co., Ltd., Tokyo, Japan). Then, the pupil was dilated with phenylephrine hydrochloride and tropicamide eye drops (Santen Pharmaceutical Co., Ltd., Osaka, Japan), and 20 nmol NMDA with or without tranylcypromine was injected into the vitreous cavity. To assess the inhibitory effect of mitogen-activated protein kinase (MAPK), 100 nmol BIRB796 was intravitreally injected at the same time of NMDA injection. The injections were performed under a microscope using a 33-gauge needle connected to a microsyringe (Ito Corporation, Shizuoka, Japan); the needle was inserted approximately 1.0 mm behind the corneal limbus. Next, either PBS (vehicle control) or 500 mM tranylcypromine (1000 nmol) mixed with 10 mM NMDA (20 nmol) in a total volume of 2.0 lL was injected into the vitreous cavity.
9
Electroretinography Assessment of Diabetic Mice
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Electroretinography was inspected at 20 weeks after the DM model was established. All mice were dark-adapted overnight in a dark chamber after pupil dilation was induced by tropicamide eye drops (Santen Pharmaceutical Co., Ltd. Kita-ku, Osaka, Japan). Anesthesia, consisting of ketamine and xylazine, was administered the next day. The mice were then placed on a heating board. The reference and ground electrodes were inserted into the palate and tail, respectively. Platinum corneal electrodes were placed on cornea of both eyes, and recombinant bovine fibroblast growth factor eye gel was applied for lubrication. Mouse ERG preparation was completed under dim red lighting in the dark chamber. Illumination intensities of 0.0004, 0.04, 4, 400, and 2000 cd•s/m2 were used to record scotopic ERG by Espion electroretinogram E2 system (Diagnosys, Lowell, MA, USA) Then, the mice were light adapted for 10 min, and photopic ERGs were recorded under an illumination intensity of 2000 cd•s/m2.
10
Hole-ICL Implantation Technique
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Each surgery was performed by the same experienced specialist (J Zhang). Before surgery, instillation of mydriasis agents (Tropicamide Eye Drops, Santen, Japan) was performed four times at 10-min intervals, then a 3-mm temporal corneal incision was made with a diamond knife and then the ICL V4c with a 0.36-mm central artificial hole (Hole ICL™) was inserted. A small amount of viscoelastic agent was injected in the anterior chamber after implanting the ICL. The four footplates of the ICL were placed on the ciliary sulcus behind the iris with two manipulators along the 180° axis. Viscoelastic agent was totally cleared using buffered salt solution. The ICL position was verified before the surgery was finished.
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