| The pupil light reflex (PLR) is pupils’ size change caused by light exposure. It is causedby the impulses of the rod cells, cone cells and intrinsically photosensitive retinal ganglioncells(ipRGCs)[1]. PLR is consists of pupils narrowing (constriction) and widening (dilation)processes and it is an important life sigal. Central nervous system and autonomic nervoussystem play an important role in the process of PLR. PLR will be weakened or disappearedif the vision system or nervous system get some lesion[10].So the measure of PLR have awidely use in some clinical and basic research works[11,12]of diagnosing and evaluating thedisease of vision system[2-5], nervous system[6,7], psychology diseases, and so on[8,9].Thepupillary movement could be measured by infra camera (infrared pupillometry). There aredifferent indexes to describing pupil movement behavior by infrared pupillometry and thedetail dynamic changes of the pupil diameter could be showed by pupil movement waves[13]. However, the measure of PLR in animal experiments is still subject to certainrestrictions due to some influencing factors such as light stimulation conditions, restriction,anesthesia methods and iris pigment. To resolve the questions above, we did this research.First, we explored the impacts of the different stimulus light color, and luminance on theC57BL/6mice pupil light reflex (PLR) dynamic analysis parameters used by the infraredpupillometry.Secondly, we compared the C57BL/6mouse PLR performed using restraint only, ketamine and/or xylazine. We also measured heart rate as other putativepharmacodynamic measure of these drugs. The thirdly, the PLR behavior andelectroretinogram were compared in wild type mouse and a retinal degeneration fast(rdf/B6) in order to explore retinal function objective evaluation method.Materials and methods1.30adult male C57BL/6mice (SPF grade, purchased from the Experimental AnimalCenter of the Fourth Military Medical University) for the pre-experimental andexperimental research.20rdf/B6mice (SPF grade, purchased by the animal barrierlaboratory of the Department of Aerospace Medicine in Fourth Military Medical University)were used.2. The PLR was measured using a pupillometry system developed by our department[14].During testing, the mouse was followed by white, red, green and blue light stimulation,each color of light given luminance order2cd/m~2,8cd/m~2and32cd/m~2,128cd/m~2,256cd/m~2. We outputed PLR curve and analysis the relevant parameters; we compared themouse PLR curve, pupil size and heart rates under restraint, ketamine, xylazine, andketamine/xylazine, respectively used by the pupillometry and electrocardiograph; Utilizedthe technique of visual electrophysiology to record the color flash ERG of C57BL/6mouseand rdf mouse and the PLR of rdf mouse under different stimulate light color andluminance was recorded by the pupillometry system too.3. SPSS (version13.0) was used for statistical analyses.Results1. The relationship between stimulus brightness and PLR dynamic analysis curveparameters of C57BL/6miceFrom2cd/m~2to256cd/m~2five light intensity levels of white, red, green and blue lightstimulates, the difference of CL was not statistically significant;CA is positive correlationwith the luminance of the white, green and blue light (P<0.01,Fwhite=7.591，Fgreen=25.756 ，Fblue=70.319), and has no significant correlation with the red light; CV is negativecorrelation with the luminance of the white, green and blue light(P<0.01,Fwhite=7.308，Fgreen=5.497，Fblue=11.651), and has no significant correlation with the red light.RV isnegative correlation with the luminance of the white, green and blue light (P<0.01,Fwhite=8.386，Fgreen=23.517，Fblue=66.556), and has no significant correlation with thered light.2. The relationship between stimulus light color and PLR dynamic analysis curveparameters of C57BL/6miceParameters of PLR curve had relation with the stimulation luminance. With increasingof brightness, the difference of CL caused by the four colors of light is not significant (P>0.05); CA caused by red light significantly less than white (P<0.05), CA caused by bluelight significantly greater than white (P<0.05), CA caused by the green and white light hadno significant difference (P>0.05); CV caused by red light significantly greater than white(P<0.05), CV caused by blue light significantly less than white (P<0.05), CV caused bygreen and white light had no significant difference (P>0.05); RV caused by red light isgreater than white (P <0.05), blue light caused by RV is less than white (P <0.05), RVcaused by green and white light was no significant difference (P>0.05).3. Effect of restraint，ketamine, xylazine and ketamine/xylazine on measures ofpupillary light reflex in male C57BL/6miceThe changes of pupil diameter during the restraint could be reduced and mice could becalm enough to be given PLR measure by giving3days to5days restraint adaption.Restraint adaption may be a good method for taking PLR measure to evaluate the functionof automatic nervous system in mouse models awake but the stress of the restraint is stillexisted. Ketamine or ketemine/xylazine could decrease initial pupil diameter（IPD）and theconstriction amplitude (CA) in the mice. Constriction latency (CL) in ketamine group andketamine/xylazine group had a tendency to prolong (P=0.016and P=0.020, respectively).Constriction velocity (CV) in ketamine group was larger than restraint group andketamine/xylazine group (P=0.008and P<0.001, respectively) and there were nosignificant difference between re-dilation velocity (RV) of pupil in the restraint group, ketamine group and xylazine group. The PLR in xylazine group could not been observed inthis study. Heart rates of the mice were reduced in ketamine group, xylazine group, andketamine/xylazine group compared with restraint group. The arrhythmia was also recordedin ketamine group but not the other groups.4. Characterization of electroretinography to chromatic light stimuli in C57BL/6mice and rdf/B6miceThe color Flash ERG of the C57BL/6mouse showed: The amplitudes of b-wave of RodERG and Max ERG caused by red light stimulation were weaker than the white, green andblue (P<0.01). The implication time of b-wave of Rod ERG and Max ERG caused by redlight stimulation were prolonged than the white, green and blue (P<0.01). The amplitudesof b-wave of Photopic ERG caused by blue light stimulation were larger than the white,green (P<0.01) and the Photopic ERG could not observed after the red light stimulates,theERG caused by the green and white light had no significant difference.The color flashERGs of the rdf/B6mouse had no waves.5. Characterization of PLR to chromatic light Stimuli in C57BL/6mice and rdf/B6miceIn the luminance of2cd/m~2white, red, green and blue light stimulation, PLR ofrdf/B6mousewere not obvious. In the luminance of30cd/m~2white, red and green lightstimulation, PLR of rdf/B6mouse were not obvious too,but the the blue light could causeobvious PLR and the constriction amplitude of the pupil were smaller than C57BL/6mice（P<0.01）. In the luminance of500cd/m~2white, red, green and blue light stimulation, PLRof rdf/B6mouse were obvious too,but the the blue light could cause obvious PLR and theconstriction amplitude of the pupil were smaller than C57BL/6mice（P<0.01）.Theconstriction amplitude caused by blue light in rdf/B6mouse was larger than white light andgreen light（P<0.01）and had no siginificant different with the C57BL/6mouse. Theconstriction amplitude caused by green light in rdf/B6mouse had no siginificant differentwith the C57BL/6mouse too, but the constriction amplitude caused by white light wasweaker than the C57BL/6mouse. The PLR caused by the red light was still not obviouseven the luminance was500cd/m~2. ConclusionsThe results suggest that:1. The PLR dynamic curve parameters of C57BL/6mice are closely related to thecolor and brightness of the light stimulus. We should select the appropriate conditionstimulus light to use the PLR test technology to evaluate the function of mouse models.2. Ketamine or ketamine/xylazine can be used for mice sedating but the influences ofketamine and xylazine on some parameters of mouse PLR needs to be paid attention to.The amplitude of spontaneous movement of pupil could be reduced and mice could becalm enough to be given PLR measure by giving3to5days restraint adaption. Ketamine,xyzaline or ketemine/xylazine will alter some parameters of the mouse PLR. Restraintadaption may be a better way for PLR measure to evaluate the automatic nervous system inmouse models. Restraint adaption or sedated by anesthetic to perform the PLR measure ofmouse should be based on research needs and the characteristics of the animal models.3. The color flash ERGs of C57BL/6mouse were different during different colorstimulate light and rdf/B6mouse had no obvious color flash ERGs waveforms. Blue andhigh-luminance stimulation light could leads to obvious PLR in rdf/B6mice. Theapplication of the pupillometry system can reflex the function of the ipRGCs in rdf/B6mice. The combination utilized of pupillometry system and visual electrophysiology can beable to more fully comprehensive assessment of visual function. |
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