Additional file 1 of Swimming exercise reverses transcriptomic changes in aging mouse lens

Supplementary Material

The retinal levels of 5-HT2AR in three groups.

<p>(A) The number of positive cells in retinal ganglion cells(RGCs) of both FDM and FLM eyes increased significantly compared with the control group (<i>p</i><0.001). (B) The average optical density of 5-HT2AR positive cells in inner nuclear layers (INL) was calculated for the three groups, respectively. In the layer of INL, the number of 5-HT2AR increased both in FDM and FLM groups, significant differences were detected among these three groups (<i>p</i><0.05) (*<i>p</i><0.05 vs control group; **<i>p</i><0.001 vs control group;^#<i>p</i><0.05 vs FDM group; all data were showed as mean±SEM).</p

The dynamic status of axial length of guinea pigs in control, FDM and FLM groups.

<p>(A) The axial lengths kept elongating in three groups with passage of time during the experimental period. The axial lengths of FDM and FLM eye were significantly longer than that of control eyes from the 2nd week(both <i>p</i><0.05). (B) Over the 8-week experiment, changes in axial lengths were greater in FDM and FLM eyes than those in control eyes (both <i>p</i><0.001) while significant difference was not found between FDM and FLM group. (*<i>p</i><0.05 vs control group; **<i>p</i><0.001 vs control group; data were expressed as mean ± SEM).</p

Effects of Constant Flickering Light on Refractive Status, 5-HT and 5-HT2A Receptor in Guinea Pigs

<div><p>Purpose</p><p>To investigate the effects of constant flickering light on refractive development, the role of serotonin (i.e.5-hydroxytryptamine, 5-HT)and 5-HT2A receptor in myopia induced by flickering light in guinea pigs.</p><p>Methods</p><p>Forty-five guinea pigs were randomly divided into three groups: control, form deprivation myopia (FDM) and flickering light induced myopia (FLM) groups(n = 15 for each group). The right eyes of the FDM group were covered with semitransparent hemispherical plastic shells serving as eye diffusers. Guinea pigs in FLM group were raised with illumination of a duty cycle of 50% at a flash frequency of 0.5Hz. The refractive status, axial length (AL), corneal radius of curvature(CRC) were measured by streak retinoscope, A-scan ultrasonography and keratometer, respectively. Ultramicroscopy images were taken by electron microscopy. The concentrations of 5-HTin the retina, vitreous body and retinal pigment epithelium (RPE) were assessed by high performance liquid chromatography, the retinal 5-HT2A receptor expression was evaluated by immunohistofluorescence and western blot.</p><p>Results</p><p>The refraction of FDM and FLM eyes became myopic from some time point (the 4th week and the 6th week, respectively) in the course of the experiment, which was indicated by significantly decreased refraction and longer AL when compared with the controls (<i>p</i><0.05). The concentrations of 5-HT in the retina, vitreous body and RPE of FDM and FLM eyes were significantly increased in comparison with those of control eyes (both <i>p</i><0.05). Similar to FDM eyes, the expression of retinal 5-HT2A receptor in FLM eyes was significantly up-regulated compared to that of control eyes (both <i>p</i><0.05). Western blot analysis showed that retinal 5-HT2A receptor level elevated less in the FLM eyes than that in the FDM eyes. Moreover, the levels of norepinephrine and epinephrine in FDM and FLM groups generally decreased when compared with control groups (all <i>p</i><0.05).</p><p>Conclusions</p><p>Constant flickering light could cause progressive myopia in guinea pigs. 5-HT and 5-HT2A receptor increased both in form deprivation myopia and flickering light induced myopia, indicating that 5-HT possibly involved in myopic development via binding to5-HT2A receptor.</p></div

Immunofluorescene photographs of retina obtained by staining 5-HT2A receptors (5-HT2AR) and nuclei.

<p>Red fluorescence represented 5-HT2AR, and blue represented cell nucleus (scale bar: 25μm). Up-regulation in the expression of 5-HT2ARweremainly found in the layer of retinal ganglion cells(RGCs), inner nuclear layers (INL) and photoreceptors after in both FDM and FLM group.</p

The Western blot results of 5-HT2A receptor expression in three different groups.

<p>The 5-HT2A receptor expression significantly increased in different degrees in FDM and FLM groups when compared with control group, in which the FDM group increased the most. (*<i>p</i><0.05 vs control group; ^#<i>p</i><0.05 vs FDM group; all data were showed as mean±SEM).</p

Corneal radius of curvature (CRC) in control, FLM and FDM groups over the 8-week experiment.

<p>(A) CRC of the right eyes in three groups throughout the examined period; Compared with control eyes, CRC of the right eyes in FDM and FLM group became greater at the 8th week (both<i>p<0</i>.<i>001</i>). (B) The CRC alterations in the right eyes of three groups by the end of the experiment; No significant difference was found between FDM and FLM groups (<i>p>0</i>.<i>05</i>), but both groups have significant difference when compared to the control group (both<i>p<0</i>.<i>001</i>). (**<i>p</i><0.001 vs control group; data were expressed as mean±SEM).</p

The concentrations of 5-HT contained in the retina, vitreous body and RPE of guinea pigs in different groups after 8 weeks’ experiment.

<p>Treatments in FDM and FLM groups resulted in a significant increase in 5-HT concentrations all three parts in comparison with corresponding part in control group (all <i>p</i><0.05). (*<i>p</i><0.05 vs control group; **<i>p</i><0.001 vs control group;^#<i>p</i><0.05 vs FDM group; data were expressed as mean ± SEM).</p

The transmission electron microscope photographs of the disc membranes in three groups.

<p>Morphologically, in comparison with control group, both FDM and FLM groups displayed several abnormalities: membrane discs of outer segment of photoreceptors were apparently disordered and the gap between membrane disc became widen, when compared with the control group (scale bar: 500nm).</p

The dynamic status and changes of refraction of guinea pigs in control, FDM and FLM groups.

<p>(A)In comparison with control group, significant myopic shift in the right eyes in FDM and FLM group appeared from the 4th week and the 6th week, respectively. (B) In the right eyes when compared with control group, significant changes in the refraction were proved in both FDM and FLM groups. (C)In comparison with control group, there was no significant myopic shift in the left eyes of FDM group whereas significant myopic shift in the left eyes of FLM group was established starting from the 6th week. (D) In the left eyes significant changes in refraction were found in FLM group when compared with either control group or FDM group. (FDM: Form-deprived myopia group; FLM: Flickering light induced myopia group; R: right eyes; L: left eyes. Error bars denote standard errors. (*<i>p</i><0.05)compared with control group; **<i>p</i><0.001 vs control group; ^#<i>p</i><0.05 compared vs FDM group; ^##<i>p</i><0.001 vs FDM group).</p