Voluntary exercise preserves visual function and reduces inflammatory response in an adult mouse model of autosomal dominant retinitis pigmentosa

Abstract

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.

Figure 1

Voluntary running wheel exercise preserved retinal function in induced I307N Rho mice. To test the effects of voluntary exercise on retinal and visual function, we performed electroretinography (ERGs) and optomotor response (OMRs) measurements at baseline, 2- and 4-weeks post light induction (a). To ensure exercise-induced retinal neuroprotection was achieved as demonstrated in our labs previously, electroretinography (ERG) recordings were performed to non-invasively measure retinal function. Representative ERG waveforms are shown from dark-adapted stimuli (2.1 log cd s/m²; b). Active induced mice had significant preservation of a-wave (c), b-wave (d) and photopic b-wave amplitudes (e) compared to inactive induced mice. a- and b-waves show rod photoreceptor and inner retinal function, respectively at 2-weeks post light induction. The photopic ERG measures cone photoreceptor function. Two-way ANOVA with Tukey’s multiple comparison analysis was performed. N = 11–13 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values are mean ± SEM. IF immunofluorescence.

Figure 2

Contrast sensitivity and spatial frequency thresholds are preserved in active induced mice compared to inactive induced mice. Optomotor response (OMR) was performed at baseline and 1- and 2- weeks post-induction to measure visual function. At 1- and 2-weeks post induction, active induced mice had significantly greater contrast sensitivity (a) and spatial frequency (b) thresholds compared to inactive induced mice. N = 11–16 per group, **p < 0.01, ***p < 0.001, ****p < 0.0001. Values are mean ± SEM.

Figure 3

Active induced I307N Rho mice maintained photoreceptor nuclei. Retinal sections from all experimental groups (a,c) were used to quantify photoreceptor nuclei present in the outer nuclear layer (ONL). Morphometric analyses were constructed by plotting the quantification of ONL nuclei and cone photoreceptors by cone arrestin labeling as a function of position in the retina relative to the optic nerve (μm) spidergram (b,d). Although photoreceptor loss was evident in both induced groups, active induced mice had consistently greater numbers of outer nuclear layers when compared to inactive induced groups. Retinal layers are as follows: outer segment (OS), inner segment (IS), outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL). N = 8 per group, 3 retinal sections quantified per animal, ****p < 0.0001. Values are mean ± SEM.

Figure 4

Cytokine expression elevated in inactive vs active mice. (a) Measured cytokines (columns, z-scored) from retinal extracts of all experimental animals (rows). (b) Partial least squares discriminant analysis (PLSDA) identified a profile of cytokines, LV1, correlated with active cases (negative) or inactive cases (positive). (c,d) LV1 separated inactive induced and uninduced cases to the right (unfilled shapes) and active induced and uninduced cases to the left (filled shapes). (e) Univariate analysis revealed significantly higher expression of KC, IP-10, and IL-13 in inactive vs active mice (*p < 0.05, unpaired t-test with Welch’s correction) and trending higher expression in inactive mice for VEGF, G-CSF, and MIG. N = 4–9 per group, each symbol in the plots represent retina from individual animals.

Figure 5

KC and IP-10 labeling localized to retinal vasculature. To determine the retinal cell type(s) expressing KC and IP-10, immunofluorescence on sagittal retinal sections from experimental groups was performed. Immunofluorescence revealed KC (a–d, green) and IP-10 (f–i, green) labeling localized to retinal vasculature as shown with co-labeling for collagen IV (a–i, red). We also observed inactive induced mice had a significant increase in both KC (e, p = 0.0110) and IP-10 (j, p = 0.0220) compared to uninduced mice. N = 6 animals per group, each symbol in the plots represents the average of three retinal sections per animal. KC and IP-10 labeling quantification are the result of positive KC and IP-10 labeling within the superior, inner and deep vascular plexi, *p < 0.05. Values are mean ± SEM.

Figure 6

Moderate negative correlation of IP-10 expression associated with retinal and visual function assessments. Heatmap analyses was used to determine significant differences between individual animals for KC and IP-10 expression, retinal function (scotopic a- and b-wave function and photopic b-wave function) and visual function (spatial frequency; SF and contrast sensitivity; CS). IP-10 expression was found to have a moderate negative correlation with retinal (a-wave r = 0.41, p = 0.030; b-wave r =  − 0.40, p = 0.036; photopic b-wave r =  − 0.43, p = 0.024) and visual (SF r =  − 0.40, p = 0.036; CS r =  − 0.39, p = 0.041) function assessments. There was no significant correlation between KC expression and retinal (a-wave r = 0.201, p = 0.304; b-wave r = 0.182, p = 0.353; photopic b-wave r = 0.127, p = 0.520) and visual (SF r = 0.011, p = 0.958; CS r = 0.151, p = 0.442) function. N = 4–9 animals per group.