Full-field scotopic electroretinography (ERG) was performed to assess the retinal function of the leptin-deficient (ob/ob) mice and the littermate controls. Animals were dark adapted overnight and anesthetized with an intraperitoneal injection of ketamine (100 mg/kg; Troy Laboratories, Sydney, Australia) and xylazine (10 mg/kg; Troy Laboratories). The pupils were dilated with 1% atropine sulfate (Bausch + Lomb, Rochester, NY). ERG was performed using a single paradigm to elicit mixed responses (rod and cone) according to previously established methodology [23 (link)]. The a- and b-wave amplitudes were measured over a stimulus intensity range of −4.4 − 1.9 log cd·s·m-2. Data are expressed as the mean wave amplitude ± standard error of the mean (SEM; µV). Statistical significance (p<0.05) was determined using two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons post-hoc test, using Prism 5 software (GraphPad, La Jolla, CA).
Atropine sulfate
Manufactured by Bausch & Lomb
Sourced in United States
Atropine sulfate is a chemical compound commonly used in ophthalmic laboratory equipment. It is a white crystalline powder that acts as a parasympatholytic agent, primarily blocking the muscarinic receptors of the parasympathetic nervous system. This compound is often utilized in various ophthalmic applications, such as pupil dilation and the treatment of certain eye conditions.
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Lab products found in correlation
Slr based photo slit lamp imaging system, by Bausch & Lomb (3 mentions)
Sl d8z slit lamp biomicroscope, by Nikon (2 mentions)
Ketamine, by Troy Laboratories (1 mentions)
Xylazine, by Troy Laboratories (1 mentions)
Prism 5, by GraphPad (1 mentions)
Phenylephrine hydrochloride, by Bioteck (1 mentions)
Tropicamide, by Bausch & Lomb (1 mentions)
Utas 2000, by LKC Technologies (1 mentions)
Phenylephrine hydrochloride, by Akorn (1 mentions)
Sliding microtome, by Leica camera (1 mentions)
Nis elements software, by Nikon (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Ds fi1, by Nikon (1 mentions)
Labchart 8, by ADInstruments (1 mentions)
Genteal lubricant eye gel, by Alcon (1 mentions)
Micron 3 imaging system, by Phoenix Pharmaceuticals (1 mentions)
Gonak, by Akorn (1 mentions)
Carprofen, by Pfizer (1 mentions)
Isoflurane, by Phoenix Pharmaceuticals (1 mentions)
Ketamine, by Phoenix Pharmaceuticals (1 mentions)
15 protocols using atropine sulfate
1
Assessing Retinal Function in Leptin-Deficient Mice
2
Light-Induced Retinal Damage in Mice
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Light damage of mice was induced as previously described. Briefly, mice were adapted in darkness overnight, and eyes were dilated with 1% atropine sulfate (Bausch & Lomb) and 10% phenylephrine hydrochloride (Paragon BioTeck). Mice were then placed in a reflective container with a cool white-light LED light source (Fancierstudio), which was placed above the container with 65,000 lux adjusted using an illuminance meter. After 6 h exposure for Cx3cr1YFP-CreER mice or 8 h for other C57BL/6J mice, the mice were returned to the housing facility with normal lighting and bred for additional five days before experiments.
3
Induced Retinal Stress in Rodents
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To induce retinal stress, we implemented a PD paradigm. The adult SD rats were placed in transparent Perspex open-top cages under a light source (COLD F2, 2×36W, IHF, Thorn Lighting, Spennymoor, United Kingdom) at 1,000 lux for 24 h, with access to food and water ad libitum [31 (link)]. The C57BL/6J mice were housed in custom-made Perspex boxes coated with a reflective interior, and exposed to 100 K lux of natural white light-emitting diode (LED) for up to 7 days, with free access to food and water [32 (link)]. Each animal was administered pupil dilator eye drops (atropine sulfate 1% w/v, Bausch and Lomb, Rochester, MN) twice daily during PD.
4
Light-Induced Retinal Degeneration in Mice
5
Characterization of bs2 Mutant Mice
6
Scotopic Electroretinography in Retinal Ischemia
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Mice were dark-adapted overnight and anesthetized using xylazine (20 mg/kg, intraperitoneally) and ketamine (80 mg/kg, intraperitoneally). Pupils were dilated with phenylephrine hydrochloride (2.5%; Akorn) and atropine sulfate (1%; Bausch & Lomb, Tampa, FL, USA). Contact lens electrodes were placed on both eyes accompanied by 2.5% hypromellose ophthalmic demulcent solution (Goniovisc; HUB Pharmaceuticals, LLC, Rancho Cucamonga, CA, USA). Full-field scotopic electroretinograms (ERGs) were recorded as described previously,47 (link) using universal testing and electrophysiologic system 2000 (UTAS-2000; LKC Technologies, Gaithersburg, MD, USA). Single white flashes (10 ms) with intensity of 2.48 cd/s/m2 were used for stimulation. No frequency filtering was used. A-wave amplitudes were measured from baseline to the a-wave trough. The b-wave amplitudes were measured from the a-wave trough to the peak of the b-wave. ERGs were recorded 24 hours before the retinal ischemia and 7 days post ischemia.
7
Histological Analysis of Mouse Eyes and Gonads
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Mouse eyes were examined with a Topcon SL-D8Z slit lamp biomicroscope with a Nikon SLR-based Photo Slit Lamp imaging system following mydriasis with 1% Atropine Sulfate (Bausch & Lomb). Eyes, brains, and testes were collected at 8 weeks of age. Eyes and testes were fixed in 4% paraformaldehyde (PFA), paraffin embedded and H&E stained as previously described [5 (link)]. Brains were fixed at 4°C for 24 h in 4% PFA followed by 30% sucrose for 24-72 hrs. Brains were then sectioned at 30 μm on a sliding microtome (Leica) and stained with DAPI to label all nuclei. Immunostaining was done with TRA54 (B-Bridge) as a primary antibody and DyLight 488 goat anti-rat (Abcam) as a secondary antibody following the manufacturer’s recommendations. PNA staining was performed utilizing the Lectin PNA-Alexa-488 conjugate (Life Technologies) according to the manufacturer’s recommendations. Slides were DAPI stained according to the manufacturer’s recommendations (Life Technologies), mounted using Fluoromount-G (Southern Biotech), and imaged using a Nikon DS-Fi1 camera on a Nikon Eclipse 80i microscope using NIS-Elements software (Nikon).
8
Assessing Retinal Function via Scotopic ERG
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Full-field scotopic ERG was performed to assess the animals’ retinal function after intravitreal injections as described previously [32 (link)]. ERG was performed using an LED-based system (FS-250A Enhanced Ganzfeld, Photometric Solutions International, Huntingdale, Australia). Briefly, mice were dark-adapted overnight, anesthetized using an intraperitoneal injection of ketamine (100 mg/kg) and xylazil (12 mg/kg), and the pupils dilated with 1% w/v atropine sulfate (Bausch and Lomb). A single- or twin-flash paradigm over a stimulus intensity range of 6.3 log cd s m−2 (range −4.4 to 1.9 log cd s m−2) was used to elicit mixed (rod and cone) or isolated cone responses, respectively. Measurements of the cone a-wave and b-wave responses were performed using Lab Chart 8 (AD Instruments, Dunedin, New Zealand).
9
Rat and mouse photoreceptor damage protocols
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For the rat PD model, animals, housed and exposed to bright (1,000 lx) light for 24 h in accordance with our previous protocols. Exposure began and ended at 09:00 a.m. on successive days. Rats were euthanized for tissue collection either immediately following PD (0 days), or after a further 3 or 7 days in cyclic dim light. Dim-reared animals were collected as non-light exposed controls for comparison.
The mouse PD model was performed following our previously established methodology (42 (link)). In brief, age-matched wild type (C57BL/6) and complement knockout animals were housed in Perspex boxes coated with a reflective interior, and exposed to 100 K lx of natural white LED for up to 7 days, with access to food and water ad libitum. Each animal was administered with pupil dilator eye drops (0.1% atropine sulfate, Bausch and Lomb, Australia) two times a day during light exposure. Animals were either euthanized or subjected to electroretinogram (ERG) recordings after 3, 5, 7 days of PD.
The mouse PD model was performed following our previously established methodology (42 (link)). In brief, age-matched wild type (C57BL/6) and complement knockout animals were housed in Perspex boxes coated with a reflective interior, and exposed to 100 K lx of natural white LED for up to 7 days, with access to food and water ad libitum. Each animal was administered with pupil dilator eye drops (0.1% atropine sulfate, Bausch and Lomb, Australia) two times a day during light exposure. Animals were either euthanized or subjected to electroretinogram (ERG) recordings after 3, 5, 7 days of PD.
10
Phenotypic Analysis of bs2 Mutant Mice
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Weights of WT (n = 8) and bs2 (n = 8) postnatal mice were measured and recorded in littermates from bs2/+ × bs2/+ crosses between P0.5 and 4 months of age. Age-matched bs2 (n = 4), AGPS-KOMP mice (n = 4), Agps-KOMP EIIa-Cre (n = 2) and control (n = 4) mice were X-ray imaged at 4 months of age. Exposures were recorded at a peak kilovoltage of 50 kVp and a charge of 0.50 mAs (milliampere seconds). The same mice were also evaluated with a Topcon SL-D8Z slit lamp biomicroscope with a Nikon SLR-based Photo Slit Lamp imaging system following mydriasis with 1% Atropine Sulfate (Bausch & Lomb). WT (n = 6) and bs2 (n = 6) testes weights were measured in age-matched pairs between 4 and 8 weeks of age. Significance for all measurements was calculated via two-tailed t-test (GraphPad), where P < 0.05 was considered significant.
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