Abstract:

It is not currently known if impaired visual function following oil exposure in early life stage zebrafishes is a downstream effect of cardiotoxicity, or if crude oil exposure independently affects vision in developing zebrafishes. To test this, cleavage stage zebrafish embryos (< 1.25 hpf) were microinjected with the microRNA miR133b to induce pericardial edema. Vision (optokinetic response) and cardiac (pericardial area) endpoints were then compared among 4 groups: controls, negative control miRNA, miR133b, and oil-exposed embryos.

Suggested Citation:

Leads, Rachel, Jason Magnuson, Justin Greer, Daniel Schlenk, and Aaron Roberts. 2020. Electroretinogram measures of visual function in oil exposed zebrafish (Danio rerio). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-8vck-ts91

Purpose:

To determine the effect of pericardial edema on visual function in developing zebrafish.

Data Parameters and Units:

Endpoints were collected for 4 groups: true controls (Control), negative control microRNA (negmiRNA), miR133b (posmiRNA), and oil-exposed (HEWAF) at 7 days post fertilization Larval fish age = 7 days post fertilization Optokinetic Response (OKR) = total number of smooth eye saccades Eye area = area of left eye measured in mm2 Eye diameter = diameter of left eye measured in mm Pericardial area = pericardial area of the left lateral view measured in mm2 Total body area = total body area of the left lateral view measured in mm2 Total length = total length of the left lateral view measured in mm Survival = Total larvae hatched alive at 7 days post fertilization Remarks: Note that the 100-um scale bar on some of the images is incorrect. The scale bar is 1 mm.

Methods:

Preparation of Water Accommodated Fractions: High energy water accommodated fractions (HEWAF) were prepared with a loading rate of 1 gallon of oil per 1 liter of egg water. Naturally weathered oil from the surface (OFS) was used in all HEWAF preparations. The oil-water mixture was blended in a Waring CB15 blender for 30 s at low speed. The resulting mixture was immediately transferred to a glass separatory funnel and was allowed to settle for 1 hour. Following the 1 hour settling period, the bottom 90% of the mixture was drained and was determined to be the 100% HEWAF. The 100% HEWAF was then diluted with egg water to a nominal 4% exposure solution. All oil exposures were conducted at a nominal concentration of 4% OFS HEWAF. Subsamples of the diluted HEWAFs were collected in 250 mL amber glass bottles and were stored at 4 degrees C until being sent to ALS Environmental (Kelso, WA) for total PAH50 analysis. Animal Rearing: Adult AB strain zebrafish were obtained from a local supplier (Fish n' Chirps Pet Center, Denton, TX) and were maintained in 10-gallon recirculating tanks at the University of North Texas. Fish were kept in 14 hours of light, 10 hours of dark cycle at 28 degrees C. Fish were fed commercial flake food twice per day. Oil Exposure: Embryonic zebrafish (< 4 hpf) were exposed to diluted (4%) high energy water accommodated fractions of naturally weathered oil from the surface (OFS) in 250 mL glass crystallizing dishes with 200 mL of exposure solution. A total of 4 replicates with 30 embryos per replicate were exposed to 4% OFS HEWAF. Exposure solutions were renewed at 24 and 48 h (TPAH50=54.7 to 60.4 ug/L). At 72 hpf, larvae were transferred to clean egg water for the duration of the experiment. Microinjections: Lyophilized miR133b and nc-miR mimics were obtained from Thermo Fisher Scientific (mirVana™ miRNA Mimic 133b, cat # 4464066, assay ID: MC10029; mirVana™ miRNA Mimic, Negative Control #1, cat #4464058). The mirVana™ miRNA Mimic 133b was labeled with Alexa Fluor 555 to confirm incorporation of miR133b using fluorescent microscopy. The miR mimics were reconstituted in nuclease free molecular grade water to a 1 mM stock solution and were stored at -20 degrees C. Immediately prior to injections, working concentrations (75 uM) of the miR mimics were prepared by diluting the stock solution in nuclease free molecular grade water. Cleavage-stage zebrafish embryos (1-8 cell stage, <1.25 hpf) were microinjected with approximately 3 nL of either 75 uM negative control microRNA (negmiRNA) or 75 uM miR133b using a motorized Eppendorf InjectMan $ and FemtoJet 4i. All embryos microinjected with miR133b were visualized using fluorescent microscopy at 4 hpf, and those that incorporated the miR133b were selected for the experiment. Morphological Assessment: At 7 days post fertilization, larvae were mounted in 4% methylcellulose in a 35 mm Petri dish for visualization using a Nikon SMZ800 stereomicroscope and images were taken of larvae in the left lateral position. The total standard length (mm), total body area (mm2), eye diameter (mm), eye area (mm2), and pericardial area (mm2) were measured using ImageJ. Optokinetic Response (OKR): Optokinetic response (OKR) was used to assess visual function in zebrafish larvae 7 days post fertilization. Larvae were mounted in 4% methylcellulose in a 35 mm Petri dish that was placed inside a circular drum containing alternating black and white vertical stripes. Larvae were visualized using a stereomicroscope, and videos of the OKR assay were recorded at 100 frames per second. The circular drum was attached to a motor that rotated the alternating stripes at 6 rpm. The stripes rotated clockwise for 60 seconds and counterclockwise for 60 seconds. The total number of smooth eye saccades was calculated as the sum of smooth eye saccades observed in the left eye and the right eye in the clockwise and counterclockwise directions. The number of total smooth eye saccades was recorded and compared among treatment groups.

Instruments:

Motorized Eppendorf InjectMan 4; Eppendorf FemtoJet 4i

Error Analysis:

Standard error

Individual Files: