ERGs were performed 4 months after the induction of diabetes (6 months of age). After overnight dark adaptation, mice were anesthetized with ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg), the cornea was anesthetized with 0.5% proparacaine hydrochloride (Medline, 17478-0263-12), and the pupils were dilated with 2.5% phenylephrine hydrochloride (Medline, 17478-0201-15). Briefly, mice were placed on the temperature-regulated heated ERG instrument (Diagnosys, Lowell, MA), and the retinal responses recorded by placing electrode on each cornea; the subdermal reference was placed on the nose, and ground needle electrodes were placed at the base of the tail56 (link). The full-field flash ERG responses from both eyes were recorded on mice dark-adapted overnight followed by light-adaptation (10 min of 30 cd/m2 light); the flash intensities were 0.01, 0.1, 0, 1, and 10 cd*s/m2 with a 2- to 5-s interstimulus interval. The amplitude of ERG a-wave was measured from the trough of the first corneal negative deflection to the pre-stimulus baseline. The amplitude of the b-wave was calculated from the a-wave amplitude to the peak of the b-wave. The c-wave was measured in response to 2.5 cd*s/m2 stimuli immediately before light adaptation. All ERG a-, b-, and c-wave amplitudes were automatically calculated by Espion V6 software (Diagnosys LLC).
Espion v6
Manufactured by Diagnosys
Sourced in United States
Espion V6 is a software application designed for data acquisition and analysis. It provides a user-friendly interface for controlling various laboratory equipment and instruments. The software's core function is to collect, record, and process data from connected devices.
Automatically generated - may contain errors
Lab products found in correlation
Hypromellose, by Akorn (4 mentions)
Celeris rodent erg device, by Diagnosys (2 mentions)
Celeris rodent electrophysiology system, by Diagnosys (2 mentions)
Tropicamide, by Henry Schein (1 mentions)
Celeris ophthalmic electrophysiology system, by Diagnosys (1 mentions)
Tropicamide, by Akorn (1 mentions)
Tropicamide ophthalmic solution, by Akorn (1 mentions)
Celeris erg system, by Diagnosys (1 mentions)
Prism v9, by GraphPad (1 mentions)
Proparacaine hydrochloride eye drops alcaine, by Alcon (1 mentions)
Tropicamide phenylephrine eye drops, by Santen (1 mentions)
Colordome stimulator, by Diagnosys (1 mentions)
Celeris apparatus, by Diagnosys (1 mentions)
14 protocols using espion v6
1
Electroretinogram Analysis in Diabetic Mice
2
Electroretinography of Photoreceptor Degeneration
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Electroretinography (ERG) was performed using the Espion e2 recording system (Diagnosys, Lowell, MA, USA) as previously described.28 (link) After overnight dark adaptation, scotopic ERG was recorded at 0.01, 10, and 32 log cd s/m2. After 10 minutes of light-adaptation, photopic function was assessed at 10, 32, and 100 log cd s/m2. The amplitudes were measured using Espion V6 software (Diagnosys, Lowell, MA, USA n = 18 for P23H and P23H/Fas-lpr, n = 23 for rd10/Fas-lpr; n = 20 for rd10, and n = 10 for C57).
3
Scotopic Electroretinography in Mice
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For scotopic ERG measurement, mice were dark adapted for 24 h prior to ERG recording. Each group consisted of four 5-week-old mice. Under dim red light, mice were anesthetized by intraperitoneal injection of a cocktail consisting of 20 mg/ml ketamine and 5 mg/ml xylazine in phosphate-buffered saline at a dose of 0.1 ml per 20 g body weight. Pupils were dilated with 1% tropicamide (Henry Schein, Melville, NY), and applied with 2.5% hypromellose (Akorn, Lake Forest, IL) to keep corneas hydrated. Contact electrodes were placed on corneas while the reference electrode needle was positioned subdermally between the ears. The a-wave and b-wave responses were recorded followed by a white light stimulus of different flash intensities (−3.3 to 1.7 log cd·s/m2). For each intensity, 3–20 recordings were made with the resting intervals for recovery from photobleaching and were averaged for the final amplitude. All ERGs were recorded with the Celeris ophthalmic electrophysiology system (Diagnosys LLC, Lowell, MA) and analyzed with Espion V6 software (Diagnosys LLC). Statistical analysis was performed with paired t test. Data are represented as means ± SD. The investigator performing ERG measurements was blinded to the mouse genotype.
4
Scotopic and Photopic ERG Recordings
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Scotopic ERG recording was performed as previously described15 (link). Before photopic ERG recordings, mice were kept in a lighted vivarium. After induction of anesthesia, pupils were dilated with 1% tropicamide (Akorn, no. 17478-102-12), and bathed with 2.5% hypromellose (Akorn, no. 9050-1) to keep corneas hydrated. A mouse was placed on a heated Diagnosys Celeris rodent ERG device (Diagnosys LCC), and the ocular electrodes and ground electrode were placed on the corneas and hind leg, respectively. To measure M-cone and S-cone function, stimulation was performed with alternating green light and UV light at increasing intensities. Green light stimulation (peak emission 544 nm, bandwidth 160 nm) had intensity increments of −0.5, 0.5, 1.5, and 2.5 log cd·s/m2. UV light stimulation (peak emission 370 nm, bandwidth 50 nm) had intensity increments of −1, 0, 1, and 2 log cd·s/m2. The responses for 20–25 stimuli were averaged together, and the a- and b-wave responses were acquired from the averaged ERG waveform. The ERGs were analyzed with the Espion V6 software (Diagnosys LLC).
5
Electroretinography Protocol for Dark-Adapted Mice
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Prior to recording, mice were dark adapted for 24 h overnight. Under a safety light, mice were anesthetized by intraperitoneal injection of a cocktail consisting of 20 mg/mL ketamine and 1.75 mg/mL xylazine in phosphate-buffered saline at a dose of 0.1 mL per 20 g body weight, and their pupils were dilated with topical administration of 1% tropicamide ophthalmic solution (Akorn; 17478-102-12) followed by 2.5% hypromellose (Akorn; 9050-1) for hydration. The mouse was placed on a heated Diagnosys Celeris rodent ERG device (Diagnosys LCC). Ocular electrodes were placed on the corneas, and the reference electrode was positioned subdermally between the ears. The eyes were stimulated with a green light (peak emission 544 nm, bandwidth ∼160 nm) stimulus of -0.3 log (cd·s/m2). The responses for 10 stimuli with an inter-stimulus interval of 10 s were averaged together, and the a- and b-wave amplitudes were acquired from the averaged ERG waveform. The ERGs were recorded with the Celeris rodent electrophysiology system (Diagnosys LLC) and analyzed with Espion V6 software (Diagnosys LLC).
6
Evaluating Retinal Function via Electroretinography
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Electroretinography (ERG) responses were detected by a Celeris ERG System (Diagnosys, Lowell, MA, USA) under dim red light to evaluate retinal function. After dark adapted overnight, mice were anesthetized as previously mentioned. Corneal ERGs were recorded from both eyes using gold wire loops mounted in a contact lens electrode (Mayo Corporation, Yamaguchi, Japan) following pupil dilation. The scotopic ERG was recorded at 0.01, 10, 32 log cd s/m2 intensity with stimulus of white light. Mice were then light-adapted for 10 min and photopic responses were recorded at 0.01, 10, 32, and 100 cd s/m2. Mice body temperature was maintained at 37 °C via a built-in heating board during the experiment. The amplitudes of a-wave and b-wave were measured by Espion V6 software (Diagnosys, Lowell, MA, USA).
7
Electroretinography in Anesthetized Mice
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Mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.), their pupils were dilated with 1% tropicamide, and then 2.5% hypromellose (Akorn) was applied to keep the corneas hydrated. Two sets of active recording electrodes were placed onto the corneas, and reference and ground electrodes were positioned subcutaneously between the ears and on the tail, respectively. The eyes were stimulated with alternating blue and green light at six different light intensities (0.1, 1, and 10 cd s/m2 for blue and 0.3, 3, and 30 cd s/m2 for green). The ERGs were recorded with the Celeris rodent electrophysiology system (Diagnosys LLC) and analyzed with Espion V6 software (Diagnosys LLC, www.diagnosysllc.com ). Repeated measures two-way ANOVA followed by Sidak's post hoc test were used for all ERG comparisons.
8
Evaluating Retinal Function in Mice via ERG
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Electroretinography (ERG) was used to measure retinal function in mice in response to full-field flash stimuli under scotopic conditions (with dark adaptation for approximately 16 h). A single-flash paradigm was used to elicit mixed (rod and cone) responses, over an intensity range of -2.0 to 1.6 log cd.s/m2 using the Celeris full-field ERG system (Diagnosys LLC, Lowell, MA, United States). Measurements of the amplitudes of the a-wave (photoreceptor activity) and b-wave (ON-bipolar and Müller cell activity) were performed as an assessment of retinal function using Espion V6 software (Diagnosys LLC).
9
Electrophysiological Recordings: Rigorous Analysis
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All ERG recordings were obtained with the Espion v6 software (Diagnosys LLC, Lowell, MA, USA). All data are expressed here as mean ± SEM. Two-way repeated measurements ANOVA followed by Šidák post hoc multiple comparisons test was performed in all statistical analysis [49 (link)]. Values of p < 0.05 were considered significant (indicated with *) unless otherwise indicated. Statistical analysis and graphs were plotted with the use of GraphPad Prism v.9.0 (GraphPad Software, San Diego, CA, USA).
10
Dark-Adapted and Light-Adapted ERG Recordings
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Before ERG testing, the mice were dark-adapted overnight and anesthetized with pentobarbital sodium (0.06 g/kg, IP). Pupils were dilated using tropicamide phenylephrine eye drops (Santen Pharmaceutical Co., Ltd., Osaka, Japan). Corneal surfaces were anesthetized with 0.5% proparacaine hydrochloride eye drops (Alcaine; Alcon, Geneva, Switzerland) and were covered with carbomer eye drops (Gerhard Mann, Chem Pharm Fabrik Gmbh) to increase conductivity and prevent drying. Espion Red and Espion v6 (Diagnosys, Lowell, MA, USA) were used to test ERGs. In dark-adapted ERGs, the flash luminance was 0.01 cd·s/m and 20 cd·s/m. In light-adapted ERGs after 5 minutes of light adaptation, the flash luminance was 20 cd·s/m.
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