研究目的
This study aims to identify which aspects of the pupil light re?ex are most in?uenced by rods and cones independently by analyzing pupil recordings from different mouse models of photoreceptor de?ciency.
研究成果
From speci?c gene defective mouse models which selectively voided the rod or cone function, we determined that mouse rod photoreceptors are highly contributing to the pupil response to blue light stimuli but also to low and medium red stimuli. We also observed that cone cells mainly drive the partial rapid dilation of the initial response to low blue light stimuli. Thus photoreceptor dysfunction can be derived from chromatic pupillometry in mouse models.
研究不足
The study was limited by the use of anesthetized mice, which may affect the pupillary movement, and by the potential effect of handling stress on the pupil light re?ex. Additionally, the study did not explore the full range of light intensities for blue light due to aversive head turn and eye closure response in mice at higher intensities.
1：Experimental Design and Method Selection:
The study used one-month-old wild type (WT), rodless (Rho(cid:2)/(cid:2)), coneless (Cnga3(cid:2)/(cid:2)), or photoreceptor less (Cnga3(cid:2)/(cid:2); Rho(cid:2)/(cid:2) or Gnat1(cid:2)/(cid:2)) mice subjected to brief red and blue light stimuli of increasing intensity. The maximal pupillary constriction amplitudes and the derivative curve of the ?rst 3 seconds were determined to describe the initial dynamic response to light. The constriction amplitude at
2：5 seconds after light termination was related to the maximal constriction amplitude to estimate the postillumination phase. Sample Selection and Data Sources:
The animals were handled in accordance with the statement of the 'Animals in Research Committee' of the Association for Research in Vision and Ophthalmology, and protocols were approved by the local institutional committee. The mice were kept at 208C under a 12-hour light/12-hour dark cycle with light on at 7 AM and were fed ad libitum.
3：List of Experimental Equipment and Materials:
The A2000 pupillometer for small rodents (Neuroptics, Inc., Irvine, CA, USA) was used to stimulate the retina by four banks of different wavelength LEDs in combination with a diffuser lens with a separate optical pathway for a single infrared camera for each eye.
4：Experimental Procedures and Operational Workflow:
The pupil was imaged through a telecentric lens so that small changes in distance between the lens and the eye would not affect magni?cation when the pupil border was in focus. The animal was maintained at a de?ned and constant distance from the camera to ensure a reproducible illumination between animals. The light stimulus was a 500-ms red (622 6 8 nm) or blue (463 6 8 nm) light of increasing intensities over almost a four-log unit range. Unilateral stimulation was performed and the pupil of the stimulated eye (direct response) was continuously recorded at 31 Hz.
5：Data Analysis Methods:
The pupil diameter was determined automatically by the Neuroptics, Inc. software. The pupil response data were exported to a worksheet. The baseline pupil diameter was determined from the mean pupil size during 500 ms before the light stimulus. All pupil sizes thereafter were converted to relative pupil diameter against the baseline (?100%). For each light stimulus, the averaged response from 6 to 15 pupil recordings (unilateral recordings of different animals) was calculated and plotted using the GraphPad Prism 5.01 software (GraphPad Software, Inc., San Diego, CA, USA).
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