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Published in final edited form as: Exp Eye Res. 2019 Aug 22;187:107773. doi: 10.1016/j.exer.2019.107773

Sex-Related Differences in the Progressive Retinal Degeneration of the rd10 Mouse

Baoqin Li a, Sylvia Gografe a, Alcira Munchow a, Miguel Lopez-Toledano b, Zhuo-Hua Pan c, Wen Shen a,*
PMCID: PMC6788786  NIHMSID: NIHMS1538801  PMID: 31445902

Abstract

The retinal degeneration 10 (rd10) mouse is a model of autosomal recessive retinitis pigmentosa (RP), a disease that causes blindness through the progressive loss of photoreceptors. This study shows evidence of sex-related differences in RP onset and progression in rd10 retinas. The disease onset was considerably earlier in the female rd10 mice than in the male rd10 mice, as evidenced by a loss of PDE6β proteins and rod-dominated electroretinogram (ERG) responses at an early age. Single photopic flash and flicker ERG responses and immunolabeling of opsin molecules were analyzed in both genders to assess the sex differences in the degeneration of cones in the RP retinas. The averaged amplitudes of cone-mediated ERG responses obtained from the females were significantly smaller than the amplitudes of the responses from the age-matched males in the late stages of the RP, suggesting that cones might degenerate faster in the female retinas as the disease progressed. The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway in the females. In addition, the male rd10 mice had heavier body weights than their female counterparts aged between postnatal (P)18 and P50 days. In summary, female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

Keywords: Sex difference, Retinitis Pigmentosa, rd10 mouse, Electroretinogram (ERG)

1. Introduction

Sex-related differences significantly impact the causes and severity of many common diseases as well as treatment outcomes; however, this important information is not well documented as a result of a lack of studies in biomedical research that thoroughly investigate how males and females differ. Currently, medical practice is less evidence-based for women than for men due to minimized variables and data stability provided by studies of males in biomedical research. Clearly, studying males exclusively provides half of the picture. Research on sex-related differences in ocular diseases appears to lag behind other studies of sex-related differences in both humans and animal models. Although gender has been reported to influence the prevalence and severity of major ocular diseases, such as age-related macular degeneration, glaucoma and diabetic retinopathy (Drance et al., 2001; Vajaranant et al., 2010; Mehlsen et al., 2011; Rudnicka et al., 2012), sex-related differences and their relationship with susceptibility to diseases are still the aspects for biomedical research that remain to be fully developed. Indeed, the effect of sex on most animal models of disease-related phenotypes has not been studied extensively. This study focused on the sex-related differences in disease onset and progression in the animal model of retinitis pigmentosa (RP).

RP is a degenerative eye disease characterized by the progressive death of photoreceptors in the outer retina, ultimately leading to visual impairment and blindness (Heckenlively, 1988; Pacione et al., 2003). The disease is genetically heterogeneous in nature; RP could be a result of one of more than a hundred different gene mutations and also could be inherited in an autosomal dominant, autosomal recessive, or X-linked pattern (Humphries et al., 1992; Farrar et al., 2002; Daiger et al., 2007). Indeed, many of those genes that are mutated in patients with RP are expressed exclusively in rod photoreceptors, and rod degeneration is typically followed by the secondary degeneration of cone photoreceptors (Hartong et al., 2006). With the progressive degeneration of photoreceptors, vision loss becomes inevitable in patients and animals with RP.

The pde6brd10 (rd10) mouse model of RP is a spontaneous missense mutation of the gene encoding the rod-specific phosphodiesterase β subunit (PDE6β), the catalytic subunit that hydrolyzes cGMP in response to light (Chang et al., 2002). The mutation does not cause any alterations in the pde6b RNA; however, the activity of PDE6β is substantially reduced by the mutation (Wang et al., 2018), subsequently resulting in the accumulation of free cGMP to open additional cGMP-gated channels. This change would lead to a constant Ca2+ influx through the cGMP-gated channels and cause rod photoreceptor death. Mutations in pde6b have also been identified in human patients with autosomal recessive RP or congenital stationary night blindness (Hartong et al., 2006). As reported in previous studies, rod degeneration in rd10 mice initially occurs at approximately postnatal (P) day 18, and cones degenerate after rods (Chang et al.,2007; Gargini et al., 2007). Because photoreceptor degeneration does not overlap with retinal development in rd10 mice, light-induced electroretinogram (ERG) responses can be recorded from the animals for several postnatal weeks. However, ERG responses in rd10 mice are never normal (Chang et al., 2002), typically showing reduced amplitudes of a- and b-waves in both dark- and light-adapted conditions. Usually, after 7 weeks of age, light-induced ERG responses are completely lost in rd10 mice due to the degeneration of most photoreceptors in the retina (Chang et al., 2007; Gargini et al., 2007). Indeed, increasing evidence of retinal remodeling in the rd10 retina has been reported, including neuronal rewiring in the second- and third-order neurons (Marc et al., 2003); however, the functional consequences and effect of sex on neural reorganization have not yet been determined.

We used the rd10 mouse, one of the most popular models for studying the pathogenesis of RP, to further illustrate the sex-related differences in disease onset and progression. Sex-related differences were quantified in the RP retinas by analyzing ERG responses and the residual levels of PDE6β and opsin proteins in male and female rd10 mice at comparable ages. We anticipate that this study will provide information about the importance of sexual dimorphism in the RP animal model.

2. Materials and Methods

All described procedures involving live animals were approved by the Institutional Animal Care and Use Committee of Florida Atlantic University. The study was conducted in compliance with federal regulations and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

2.1. Animals

The wild-type C57BL/6 mice and the rd10 (B6.CXB1-Pde6brd10/J) mouse strain on a C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, ME). Both strains were bred in house to produce the pups needed for the study. Monogamous breeder pairs of each strain and their offspring were housed in regular static microisolator cages under climate-controlled conditions, provided food and tap water ad libitum, and Nestlets as enrichment. The light cycle for breeder pairs was set at a 12:12 h dim light:dark cycle. Once pups were born, these cages were transferred to a dark cycle only using red light for husbandry duties (i.e., 15 min health checks daily and 1 h cage change per week) to mimic a dark condition for the mice (cf. Cronin et al., 2012)

2.2. Corneal electroretinography (ERG)

Retinal function was examined by recording ERGs from male and female mice, for a total of 91 rd10 mice and 10 wild-type C57BL mice aged from P18 to P60 days, and six to eight male and female mice per age group were analyzed. Mouse ERGs were recorded using protocols modified from procedures described in our previous study (Yang et al., 2015). Briefly, mice were transported in a covered cage to the darkroom before ERG recording and allowed to acclimate. The animals were anesthetized through intraperitoneal injections of a mixture of ketamine (70 mg/kg) and xylazine (10 mg/kg), pupils were dilated with a single drop of a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride, and animals were placed on a heating pad to avoid hypothermia. The corneal electrode was a custom-made Burian-Allen electrode designed for use with mice; a reference electrode was placed in the mouth, and a ground electrode was inserted subcutaneously near the tail. ERG recordings were conducted with mice mounted on an anti-vibration table in the dark in a shielded Faraday cage to minimize mechanical and electromagnetic noise.

A differential amplifier (Grass Instrument Co.) was used to record ERG responses, which were bandpass filtered between 0.1 and 1 kHz for single-flash responses and 0.3 and 300 Hz for flicker response, and responses were digitized at 2 kHz with a PowerLab data acquisition device (AD Instruments). Three to four light-evoked responses were averaged to increase the signal-to-noise ratio.

A white LED stimulus was used as a scotopic and mesopic light stimulus to generate ERG responses in 4 flashes for which the luminance at the cornea was approximately (log cds/m2) −2, −1.5, −1 and −0.5 (with 50-ms flashes and 20 s intrastimulus interval). A Xenon photostimulator (model PS-22, Grass Instrument Inc., Quincy, MA) was used to deliver photopic stimulation in 10-ms flashes of white light whose luminance at the cornea was approximately 1 to 3 log cd-s/m2. The stimuli irradiances were calibrated with a photometer (UDT S370 Optometer, UDT Instruments, CA). Rod-mediated ERG responses with a minimum contribution from cones were referred as scotopic ERG responses. The maximal scotopic responses were used to evaluate the maximal function of the rods in the retina. Photopic ERG responses were evoked by high-intensity stimuli that activated both rods and cones.

ERGs were analyzed according to standard practice and our previous report on mouse preparations (Yang et al., 2015; Marmor et al., 2009). For the single flash ERG response, the amplitude of the a-wave was measured from the prestimulus baseline to the most negative trough. The amplitude of the b-wave was measured from the trough of the a-wave to the most positive peak of the response, or if no a-wave was present, from the baseline to the b-wave peak. The average amplitudes of a- and b-waves in ERGs from male and female mice were plotted as bar graphs with markers. Errors are reported as standard errors of the means. The standard measurements for the flicker ERG responses were the peak-to-trough amplitude. The fundamental and secondary harmonic components in the flicker ERGs were produced by a Fast Fourier Transform (FFT) analysis. Comparisons between male and female groups were evaluated using (1) Student’s t-test for normally distributed data or (2) the Mann-Whitney U test for skewed data. A probability of p < 0.05 was considered statistically significant. Statistical analyses and curve fitting were performed with GraphPad Prism (GraphPad Software Inc.) and LabChart (AD Instruments).

2.3. Western blot analysis

After ERG recordings, the retinal tissues were extracted, whole retinal tissues were lysed in a 2× Laemmli buffer, and the total protein contents were obtained from the homogenate in the buffer solution. The solution was heated for 10 min at 95°C, centrifuged at 10,000 rpm for 5 min at room temperature, and the supernatant was collected. Equal amounts of samples were loaded into each lane of 10% Tris-glycine polyacrylamide gels (Bio-Rad), and retinal proteins were separated by electrophoresis at 100 V for 1.5 h. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare Bio-Sciences Corp.) and were probed following the Li-COR fluorescent Western blot protocol. Briefly, the PVDF membranes was immersed in the blocking solution (Li-COR) for 1 h at room temperature, and then immersed in phosphate-buffered saline containing 0.1% Tween (PBS-T) and an anti-PDE6β antibody (Invitrogen; 1:5,000) for 2 h at room temperature. Membranes were washed with PBS-T 3 times for 15 min each, and then incubated for 45 min with an IRDye 800CW goat anti-rabbit IgG secondary antibody (Li-COR; 1:10,000). After further washes with PBS-T, positively labeled bands were detected and captured using the Li-COR Odyssey scanning system.

Image Studio Lite software was used to quantitatively analyze the levels of target proteins relative to total proteins stained with REVERT Total Protein Stain Kit (Li-COR). The signal of the total protein stain was measured by applying a rectangular area at the selected range of molecular weights between 40-110 KDa. Densitometry of single bands of target proteins was performed in a rectangular area that was manually selected and adjusted to contain all detectable signal for a band, and background was subtracted using the software. Relative quantification was achieved by normalizing each target protein band to the value of total proteins, and then the ratio of the two was calculated using Microsoft Excel. All linear ranges of quantification were obtained by graphing the integrated intensities of serial dilutions and choosing a range of dilutions with the highest linearity represented by the least-squares analysis.

Data were collected into Microsoft Excel spreadsheets and analyzed using GraphPad Prism software. Unless indicated otherwise, the data presented in bar graphs represent the means ± SEM. For all analyses, P < 0.05 was considered significant. Individual statistical tests used for each experiment are described in detail in the Results or Figure Legends as appropriate.

2.4. Immunocytochemistry

After ERG recordings, the retinal tissues were prepared for immunocytochemical staining. Freshly enucleated eyes were fixed for 20 min with a phosphate-buffered saline (PBS) solution containing 4% paraformaldehyde. After removing the cornea and lens, the eyecup remained intact for further processing. It was then placed in the fixative for another 15 min, dehydrated in graded sucrose solutions (10%, 15%, 20% and 30%), and immersed in 30% sucrose overnight at 4°C. The dehydrated eyecups were embedded in OCT compound (Ted Pella), frozen overnight, and then sectioned at 14 μm on a cryostat. Frozen sections were collected on slides, air dried, and stored at −80°C.

For antibody labeling, sections were rinsed with PBS plus 0.1% Tween (PBST) containing 0.3% Triton-X (PBST-T), and then treated with a blocking solution consisting of 10% normal goat serum in PBST-T. Sections were then incubated with primary anti-S-opsin (Millipore; 1:1,000) and anti-M-opsin (Millipore; 1:1,000) antibodies diluted in a mixture containing 3% goat serum in PBST-T for 2 h at room temperature. Negative controls were performed with the same solutions, but without the primary antibody. After three 15 min washes with PBST-T, sections were incubated with a 1:2,000 dilution of Cy-3 conjugated secondary antibody (Jackson ImmunoResearch) for 40 min at room temperature. Sections were subsequently rinsed with PBST, mounted with DAPI in Vectashield mounting medium (Vector Laboratories) and viewed with a confocal laser-scanning microscope (LMS 700, Zeiss). Images were acquired with 40× oil-immersion objectives and processed with the Zeiss Microscope Zen Software.

3. Results

3.1. Sex-specific difference in the body weights of rd10 mice

We examined body weights of the animals in different age groups between P18 and P50, the critical period for RP development and progression, to evaluate the body growth of the newborn rd10 mice raised in dim red light. The histograms in Figure 1 present the average body weights of the male and female rd10 mice at the ages of P18, P25, P30-32, P36-38, P40-43 and P50. The mean values for males/females (g) were 15.4/13.2, 17.8/14.4, 20.6/15.6, 23.4/18.4, 24.3/19.8 and 25/20.1, respectively. Overall, the body weights of the female rd10 mice were approximately 17-25% less than their age-matched males. According to the information from the Jackson Laboratory website, the average body weights for C57BL/6J mice, the wild-type control, are approximately 9.7 ± 1.9 (males) and 9.3 ± 1.7 (females) in the postnatal third week; by postnatal week seven, the average body weights increased to 22.9 ± 1.5 (males) and 18.2 ± 1.1 (females), respectively. Based on the information for the wild-type mice, the body weights of the rd10 mice of both sexes are considered slightly heavier, but still in a normal range for ages between P18 and P50. These animals appeared healthy and a compromised well-being was not observed, although the animals were raised in nearly dark conditions.

Figure 1.

Figure 1.

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Average body weights measured from male and female rd10 mice aged between postnatal 18 and 50 days, showing a difference in body weight between the males and females. The error bars indicate standard errors of the means.

3.2. Sex-related difference in the onset of photoreceptor degeneration

The functional loss of photoreceptors in the early stage of RP in rd10 mice was detected in ERG responses from P18 animals. A sequence of ERG responses from the dark-adapted animals was generated by a single light flash delivering a series of intensities of light (log cd-s/m2): −2, −1.5, −1, −0.5, 1 and 1.5. The lowest intensity of light (−2 log cd-s/m2) was used to generate rod-dominated, scotopic ERG responses, while all the higher intensities of light generated ERG responses mediated by both rods and cones. Figure 2A shows an example of ERG responses obtained from a male rd10 mouse and a female rd10 mouse at P18, and a sequence of ERG responses was also obtained from age-matched wild-type C57BL male and female mice (Fig. 2B). Two principal components of an ERG response, the cornea-negative a-wave and -positive b-wave (see the green arrows in Fig. 2B), are derived from photoreceptor outer segments and bipolar cells, respectively. Compared to the wild-type control, the ERG responses of the rd10 mice showed reduced amplitudes of b-waves and substantially attenuated a-waves (Fig. 2A), consistent with a previous study (Chang et al., 2007; Gargini et al., 2007). Sequences of ERG responses were obtained from 7 pairs of male and female rd10 mice at P18; in general, the a-wave was absent following the scotopic and mesopic light stimulation and was Only present in the photopic stimulation (1.5 log cd-s/m2), but the amplitudes of a-waves were decreased to 10-15% of the wild-type control (data not shown). Therefore, we measured the amplitudes of b-wave and plotted the data in Fig. 2C. Notably, the rod-mediated ERG response (evoked by −2 log cd-s/m2) was present in all tested male rd10 mice (n = 7), but the response was markedly reduced or absent in all tested female rd10 mice (Fig. 2C, also see the dashed outline in Fig. 2A). The amplitudes of b-waves were also measured and plotted from 4 female and 3 male wild-type mice, rod-mediated b-waves were present in both males and females, and the mean amplitudes of b-waves showed no significant difference between males and females (Figs. 2D and 2F). For rd10 mice, significantly smaller mean amplitudes of b-waves elicited by the low light intensities, −2 and −1.5 (log cd-s/m2), were observed in females than in males (p<0.05, Fig. 2E). The mean amplitudes of b-waves generated by the higher light intensities, −1, −0.5, 1, and 1.5 (log cd-s/m2), showed no significant differences between the males and females (p >0.05). The mean amplitudes of a-waves generated by the photopic light intensity (1.5 log cd-s/m2) were 12.9+4.2μV (n=7) and 13.2+4.5μV (n=7) for the males and females, respectively, also showed no significant difference between the males and females. The female rd10 mice lost rod-dominated ERG responses earlier than their male counterparts at the early stage of RP.

Figure 2.

Figure 2.

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Functional loss of photoreceptors in rd10 mice at the early stage of RP. (A and B) Dark-adapted ERG responses obtained from the male and female rd10 mice and wild-type controls at P18 that were elicited by a single flash (100ms) at a series of intensities, as indicated. The dashed box in (A) indicates rod-dominated ERG responses recorded from the rd10 mice. (C and D) Amplitudes of b-waves measured from each of the tested rd10 and wild-type mice. (E) The mean values of b-wave amplitudes from a sequence of ERG responses averaged from 7 pairs of male and female rd10 mice at P18. (* p<0.05) and from wild-type C57BL females and males (F).

Rod degeneration in the RP retinas of rd10 mice was further assessed using Western blotting by measuring the levels of the PDE6β protein , because PDE6β proteins are selectively expressed in the outer segments of rod photoreceptors. As the single mutation of the pde6b gene in rd10 mouse does not reduce protein expression (Wang et al., 2018), existing PDE6β proteins can be used to quantify the number of living rods in the RP retinas. Intact retinas were excised from the male and female animals after ERG recordings and prepared for Western blot analysis. Figure 3A (left panel) shows an example of the immunoblotting results for the PDE6β protein at the expected molecular weight of 98 kDa in the retinal samples from P18 rd10 and wild-type mice. The total protein level in each lane was detected with the REVERT total protein staining kit, serving as a loading and normalization control (see the right panel, Fig. 3A). Weak PDE6β protein bands were detected in the samples from the RP retinas, indicating that a significant number of rods had degenerated in the rd10 mice at P18. Differences in the levels of the PDE6β protein in the females and males were determined by using densitometry, showing decreases in the levels of the PDE6β protein in the female and male RP retinas to approximately 7.5% and 25% of the wild-type control, respectively (n = 4, p<0.05; Fig. 3B). Thus, rod degeneration was more severe in the female RP retinas, consistent with the results from ERGs. Apparently, the onset of rod degeneration in the female rd10 mice occurred earlier and was more prominent than in the male mice.

Figure 3.

Figure 3.

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Western blot analysis of levels of the PDE6β protein in the RP and control retinas from P18 animals. (A) Major bands for the PDE6β protein at a molecular weight of 98 kDa detected in the retinal samples from the female (F) and male (M) rd10 and wild-type mice (see the left panel). The original membrane was stained with the REVERT Total Protein Staining kit and shows multiple protein bands at different molecular weights (see the right panel); total protein staining served as a loading and normalization control. (B) Densitometric quantification of levels of the PDE6β protein in the samples from the rd10 and WT mice. The numbers on the top of each bar represent the average densitometric levels of the PDE6β protein relative to the levels of the total proteins (n=4).

By P25, the rod-dominated ERG response was undetectable in the rd10 mice (dashed outline, Fig. 4A) compared to the wild-type control (right panel, Fig. 4A). Similar results were obtained from 8 male and 7 female rd10 mice at this age. Furthermore, Western blots failed to detect a PDE6β protein in the retinal samples collected from the rd10 mice after ERG recordings, as shown in Figure 4B. Based on our results, the rods in the RP retinas had degenerated by P25. At this age, the light-evoked ERG responses might be predominantly derived from cones and their On-bipolar cells in the rd10 mice.

Figure 4.

Figure 4.

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Detection of the loss of rod function and vision from age-matched male and female rd10 mice. (A) Representative typical single-flash ERG responses from the male and female rd10 mice at P25 showing the loss of rod-mediated ERG response (dashed outline) compared to wild-type control (left panel). (B) Immunoblot showing the loss of the phototransduction protein PDE6β in the RP retinas from the rd10 mice at P25.

3.3. Sex-related difference in RP progression after rods degenerated

The effects of sex on the progressive loss of cone function in the RP retinas were studied in the animals aged from P25 to P53, the time period at which the animals progressively lose vision. We examined ERG responses from these animals using a single photic stimulus with an intensity of 1 log cd-s/m2. Figure 5A illustrates typical ERG responses obtained from the female and male rd10 mice at ages of P25, P32, P39, P46 and P53, showing that the amplitudes of b-waves decreased over time. Therefore, cone functions were gradually decreasing during the progression of RP. The experiments were repeated in 6 to 8 mice of different sexes in each age group. The amplitudes of cone-mediated b-waves were measured from each animal and plotted against the postnatal ages, as shown in Figure 5B. Figure 5C shows the mean amplitudes and results of the statistical analysis; significant differences in the mean amplitudes of b-waves were not observed between the males and females at P25 (p >0.05) and P32 (p>0.05). Conversely, sex-specific differences in b-waves increased at the ages of P38-39 (p <0.05) and P44-46 (p <0.05) in the late stages of the RP; the reduction in the b-wave amplitude occurred more rapidly in the females when cone degeneration was accelerated in both sexes. By the age of P53, ERG responses were undetectable in both sexes of the rd10 mice.

Figure 5.

Figure 5.

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Decrease in cone-mediated ERG responses in the retinas from male and female rd10 mice with advanced RP. (A) Example of a single flash with an intensity of 1 log cd s/m2 that generated ERG responses from rd10 mice aged from P25 to P53. Cone functions were failing over time. (B) Amplitudes of b-waves measured from tested rd10 mice. (C) The values on the top of the bar graphs illustrate the mean amplitudes for b-waves in ERG responses from the males and females at different ages, as indicated. (* p<0.05).

Immunocytochemistry for cone opsin highlights the substantial degeneration of cones in the RP retinas from the animals aged P39, P46 and P53. Figure 6A shows examples of S- and M-cone outer segments labeled with specific antibodies against S-opsin and M-opsin in retinal sections from ventral and dorsal part of the retinas of rd10 mice (P39 and P46), and wild-type mice (P60). At P39, the M-cone outer segments of the RP retinas appeared shorter than those of the control in both dorsal and ventral regions of the retinas, indicating morphological changes in the cones of the RP retinas from both sexes. However, weaker signals for anti-S-opsin labeling were detected in the ventral part of the RP retinas from the male rd10 mice; no positive labeling was present in the RP retina from the female mice at P39 (middle panel, Fig. 6A), indicating that S-cone function was lost in the females even before P39. At P46, anti-M-opsin labeling was reduced in both dorsal and ventral parts of the RP retina; the reduction was more noticeable in the female rd10 mice. By P46, S-cones had degenerated in the RP retinas from both sexes, and no positive labeling was present in the retinal sections (left panel, Fig. 6A). Based on these results, S-cones degenerated earlier than M-cones during RP progression; additionally, more substantial S-cone degeneration was observed in the females with RP. At P53, no positive labeling for the opsin molecules was present in the RP retinas (Fig. 6C), indicating vision loss in the rd10 mice, consistent with the results of loss of ERG response at P53 shown in Figure 5.

Figure 6.

Figure 6.

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Loss of cones in rd10 mice at the late stages of RP. (A) The anti-M-opsin and anti-S-opsin labeling (red) of cone outer segments and DAPI counterstaining of the cell nuclei (blue) in retinal dorsal and ventral sections from the rd10 mice at P39 and P46. The M-opsin labeling decreased from P39 to P46, indicating a loss of cone functions over time. The S-opsin labeling was only present in the RP retinal sections from the male rd10 mice at P39. (B) The control retinal sections from mice at an age of P60 display robust staining in cone outer segments (left panel). (C) At P53, both M- and S-opsin labeling were undetectable in the RP retinal sections.

3.4. Sex-specific effect on cone-mediated temporal properties in the RP retina

The cone-mediated temporal properties of the RP retinas were further studied by recording ERG responses generated by a flickering light stimulus with various frequencies. We evaluated flicker ERG responses using frequency-response and harmonic analyses to identify if sex differentially affects the On- and Off-pathways in the RP retinas. The use of different flickering frequencies to differentiate On- and Off-components in ERG responses has been reported previously (Tanimoto et al., 2015). In a recent study using transgenic mice, Tanimoto et al. found that the temporal responses from the cone-mediated On-pathway are more sensitive to flickering frequencies between 5-15 Hz; by increasing the flickering frequency, the major temporal responses switched from the On-pathway to the Off-pathway (Krishna et al., 2002). This approach enabled us to characterize the degrees of debilitation of the RP-related changes in retinal signal processing.

Flicker ERG responses from the rd10 mice were generated by a photopic stimulus (1.5 log cd-s/m2) at stimulus frequencies ranging from 1 to 30 Hz. Figure 7A shows representative data for flicker ERG responses obtained from a male and female rd10 mouse at P36 when cone degeneration started. Enlarged views of responses from a 1-second portion at the beginning of the ERG responses at each of the stimulus frequencies, 5, 10, 15, 20 and 25 Hz, are shown in Figure 7B. The figure depicts the waveforms of cone-mediated ERG responses from On- and Off-pathways separated at the temporal frequency of 15 Hz. The amplitudes of flicker ERG responses were measured from six male and five female rd10 mice at P36 and P37 and plotted against the stimulus frequencies. Figure 7C shows the summarized frequency and response curves, indicating that the mean amplitudes of flicker ERG responses peaked at approximately 10 Hz and then steadily decreased as the stimulus frequency was further increased to preferentially activate the Off-pathway. However, at the low stimulus frequencies (<15 Hz), the mean amplitudes recorded from the female rd10 mice were noticeably smaller than their male counterparts with statistical significance (p <0.001), and the difference was less noticeable at the higher stimulus frequencies (>15 Hz). Thus, accelerated cone degeneration in the female rd10 mice might cause a more severe impairment of the On-pathway than the Off-pathway at P36-P37.

Figure 7.

Figure 7.

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Flicker light ERG responses recorded from rd10 and wild-type mice. (A) Typical flicker ERG responses recorded from male and female rd10 mice that were stimulated with a series of stimulus frequencies ranging from 1 to 30 (Hz) with an intensity of 1.5 log cd s/m2. (B) Extended time scale of (A) at frequencies of 5, 10, 15, 20 and 25 (Hz); each wave represents a 1 s segment from the beginning of the ERG responses. The stimulus frequency patterns are superimposed in gray. (C) The mean amplitude and frequency response curves averaged from females (n=5) and males (n=6) of rd10 mice at P36 and P37, and the wild-type control at P60. (D) FFT analysis of the mean amplitudes of fundamental (F) and 2nd harmonic (2F) responses and the percentage ratio of these two responses as a function of stimulus frequency obtained from the same group of mice, as well as a light-adapted wild-type C57BL mouse.

As a control, flicker ERG responses were also recorded from wild-type C57BL mice at P60 under background light to suppress rod responses. The frequency and response curves did not reveal significant sex-related differences in the control mice (Fig. 7C, black and gray traces).

Figure 7D illustrates the power spectra density of the amplitude and frequency function of fundamental (F) and synchronized 2nd harmonic (2F) components presented in Fig. 7C, which were derived from the Fast Fourier Transform (FFT) analysis. As a control, F and 2F responses were also obtained from a wild-type C57BL mouse (see the black traces in Fig. 7D). The F and 2F curves shifted in the rd10 retinas compared to the control. The properties of the fundamental and 2nd harmonic components in flicker ERG responses have been described as linear- and nonlinear-response components of retinal functions for synaptic gain control (Krishna et al.,2002; Seeliger et al., 2011). In the rd10 retinas, the amplitude spectra of the flicker ERG responses were dominated by the fundamental response (see the left panel, Fig. 7D); the 2nd harmonic component was most prominent at 5 Hz and gradually reduced as the stimulus frequency increased (see the middle panel, Fig. 7D). Thus, the 2nd harmonic response made a substantial contribution to the On-pathway but had a lesser contribution to the Off-pathway-mediated ERG response in both sexes of the rd10 mice. However, power spectrum amplitudes of both fundamental and 2nd harmonic responses were smaller in the RP retinas from the females than their male counterparts, but the difference became indistinguishable at 30 Hz (see the left and middle panels, Fig. 7D). The relative proportion of the 2nd harmonic response was normalized to the amplitudes of the fundamental response (see the right panel, Fig. 7D). At the low frequencies of 3, 8 and 10 Hz, the 2nd harmonic response contributed approximately 54%, 37% and 35% to the flicker ERG response, respectively; at the high stimulus frequency (>15 Hz), the second harmonic contributed a small fraction (<10%) to the flicker ERG responses in the RP retinas in both sexes. These results clearly indicate that the ERG responses elicited at low temporal frequencies display a much greater nonlinearity than the responses elicited with high-frequency stimuli. In summary, the flicker ERG data suggested that cone-mediated temporal responses from On- and Off-pathways in the RP retinas were still functioning, even in the late stages of the disease. Faster degeneration of cones in the female rd10 retinas at P36 and P37 caused a more substantial decrease in the On-pathway-mediated response (Fig. 7C). Due to the RP-mediated degeneration, both linear and nonlinear responses were altered in the rd10 retinal circuit.

4. Discussion

This study quantified sex-related differences in disease onset and progression in the popular RP model. We studied the RP degeneration in rd10 animals aged between P18 and P53 and provide evidence that sexual dimorphism plays a role in the onset and progression of RP. The female rd10 mice may have an earlier onset of rod degeneration compared to their male counterparts, which was supported by the data illustrating the losses of rod-dominated ERG responses and PDE6β protein at P18. Moreover, the sex-related difference in the RP retinas seemed to be comparable for the mice aged P25 to P32, the time at which the animals were undergoing puberty and sexual maturation, and the average amplitudes of b-waves measured from both genders were not significantly different (Fig. 5B). In the late stages of RP, however, cones degenerated faster in the female mice than in the males. In summary, the analyzed data illustrate the differences in RP progression between male and female rd10 mice, and the results have potential implications for future animal experiments and clinical studies on retinal degenerative diseases.

Although little is known about sex-related difference in ocular disease, sex hormones, e.g., estrogen, progesterone and testosterone, may play important roles in regulating the majority of ocular diseases, because these hormones are key regulators of vascular tone and the oxygen supply in retina and choroid (Thompson and Khalil, 2003). Indeed, estrogen stimulates gene expression of the pro-inflammatory cytokines and matrix proteins that contribute to ocular surface inflammation in dry eye syndromes in females (Suzuki and Sullivan, 2005; Chaychi et al., 2015). Again, the female gender has been identified as a risk factor for the onset and progression of normal tension glaucoma because the disease tends to occur more frequently in women Drance et al., 2001). Moreover, the female sex is a risk factor for age-related macular degeneration (Rudnicka et al., 2012; Smith et al., 1997; Friedman et al., 2004). Alternatively, diabetic retinopathy tends to occur more frequently in men (Mehlsen et al., 2011). Another study also reported a higher prevalence of primary open-angle glaucoma in men (Rudnicka et al.,2006). According to recent studies, the differences in the anatomical structure of the ocular chambers between males and females may also be a factor related to the prevalence and causes of ocular diseases (Schmid et al., 2015). Furthermore, sex-related differences in retinal gene expression have also been observed in male and female C57BL mice (Du et al., 2017). In this study, we did not conclusively determine whether the sex-related differences in the RP mouse were mediated by sex hormones, chromosomal differences or anatomical structures. Additionally, a previous study of retinal degeneration (neuronal ceroid lipofuscinosis) in a mouse model reported higher retinal cell death, as measured by TUNEL and caspase-3 activation, in the females that was directly correlated with a higher oxidation rate in the female mouse retinas and suggested a key role for the differences in oxidative stress management between sexes (Guarneri et al., 2004). These results might be an alternative explanation for the increased susceptibility of the female rd10 mice to photoreceptor degeneration in the RP retina.

Again, the rd10 mice mimic typical human RP pathology, and the purpose of this study is to provide a comprehensive analysis of sex-related differences in the diseased animal model. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

Highlights.

  • The onset of rod degeneration was considerably earlier in the female rd10 mice than in the male rd10 mice in the retinitis pigmentosa (RP) disease model.

  • As the disease progressed, cones degenerated faster in the female’s retinas.

  • The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway.

  • The female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP.

Acknowledgement

This work was supported by a Pilot Award from the Brain Institute at Florida Atlantic University (W.S.); NIH R01 grant EY14161 (W.S.) and NSF research grant IOS-1021646 (W.S.).

Footnotes

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