Identification of S- and M/L-cones in wild-type and Rd1 retinas
S- and M/L-cones were identified by immunodetection using well-characterised antibodies [15, 19]. In WT retinas, each opsin was principally associated with the outer segment (Additional file 1). Lower intensity labeling of the cone somata and synaptic terminal was also detectable, which was stronger in S-opsin+ cones than in M/L opsin+ cones. Previous studies have demonstrated that in Rd1 mice, cone outer segments degenerate prior to death of the cell body, resulting in ectopic redistribution of opsins to the surviving cell body [8]. Thus, in Rd1 retinas, cones with intact outer segments, as well as cones devoid of outer segments, are both detectable. In the present study, cone cell bodies were identified by their wide ovoid morphology and labeled with moderate fluorescent intensity, whilst outer segments were identified by their narrow bundle-like appearance and labeled with high fluorescent intensity (Additional file 2). These different cellular structures were analysed and quantified separately using image thresholding (Additional file 2). Ortin-Martinez et al [15] noted that reliable quantification of cone segments in wholemounts depends upon meticulous removal of the RPE without damaging the delicate outer segments. Retinas used in this study were successfully dissected from the RPE, however, it is possible that this was not always the case. Unlike a healthy retina from a wild-type mouse, Rd1 retinas display non-homogeneous cone degeneration. It is therefore possible that any apparent heterogeneous degeneration may be partly artefactual in nature.
Spatio-temporal characterisation of S- and M/L-cone degeneration in Rd1 retinas
Prior to examination of cone degeneration, we verified the timing of rod photoreceptor degeneration in our cohort of Rd1 mice by immunolabelling for rhodopsin using the well-charactersied antibody clone RET-P1. The results showed that rod death is an early phenotypic event. By P21, only a very sparse population of rods were still evident (Additional file 3), which had been completely lost by P28. The results are in agreement with the large volume of research conducted on Rd1 mice [2].
Our results showed a homogeneous distribution of M/L-opsin+ cones throughout the Rd1 retina at P14, whilst there was a gradient of S-opsin+ cones, from a relatively sparse population in the superior retina to a high density of cones in the inferior retina (Figs. 1-3). These results are in agreement with previous studies that examined the distribution of cones in the Rd1 retina at a very early stage of degeneration [8, 9]. At P14, the earliest time point analysed, the vast majority of S-opsin+ and M/L-opsin+ cones had preserved outer segments, but the segments were typically misshapen or swollen in appearance, whilst ectopic redistribution of opsins to the surviving cell bodies had commenced to a variable degree (Fig. 3; Additional files 4 and 5). The high concentration of intensely-labeled outer segments at P14 made cone cell body identification in wholemounts problematic. By P21, a significant proportion of outer segments had degenerated (Fig. 3; Additional files 4 and 5). Therefore, cone cell body density was only measured from P21.
Quantification of the temporal progression of S-opsin+ and M/L-opsin+ cone degeneration in wholemounts revealed a striking difference between central and peripheral areas of the retina, and, between outer segments and cell bodies. For both S-opsin+ and M/L-opsin+ cells, cone outer segment degeneration in the central retina was effectively complete by P21 (Figs. 2, 4A, B). In contrast, outer segment degeneration in the peripheral retina was more gradual, albeit that greater than 90% of S-opsin+ and M/L-opsin+ outer segments had still degenerated by P45 (Figs. 2, 4A, B). Prior to P30, M/L-opsin+ segments in the peripheral retina appeared somewhat more preserved than S-opsin+ segments (Fig. 4A, B). With regard to cell bodies, there was likewise a more rapid degeneration of both S-opsin+ and M/L-opsin+ cones in the central retina than in the peripheral retina (Figs. 2, 4C, D). For example, relative to P21, there was only 25% survival of S-opsin+ cones in the central retina, but approximately 75% remaining in the peripheral retina (Fig. 4C). The overall kinetics of S-opsin+ and M/L-opsin+ cone cell body loss in the central and peripheral retina were similar (Fig. 4C, D), but there was a marked disparity in the spatial pattern of degeneration of each cone type. There was hemispheric asymmetry in the rate of S-opsin+ and M/L-opsin+ cone degeneration; thus, S-opsin+ cones were remarkably well preserved in the inferior retina relative to the superior retina (Figs. 1-3, 5A), whilst M/L-opsin+ cones were much more resilient to degeneration in the superior relative to the inferior retina (Figs. 1-3, 5B). The disparity became more pronounced as cone degeneration progressed (Figs. 1-3, 5A, B). By P300, small numbers of M/L-opsin+ cones survived in the superior peripheral retina, while a small population of S-opsin+ cones remained in the peripheral inferior retina (Figs. 1, 2, 5A, B). Loss of S-opsin+ and M/L-opsin+ cone cell bodies in the nasal and temporal quadrants of the retina displayed similar kinetics to each other (Fig. 5C, D).
To impart perspective on the opsin protein results, we utilised qPCR to examine the levels of M/L- and S-opsin mRNAs in Rd1 retinas (normalised to a pool of two reference genes). Relative to WT retinas, the levels of both S-opsin and M/L-opsin mRNAs were lower by P14, albeit the differences did not reach significance at this time (P=0.43; P=0.56, respectively; Fig. 6). Thereafter, the level of M/L-opsin mRNA decreased in a linear fashion from P14 until P90, at which point it was 16% of the WT level (Fig. 6A). The amount of S-opsin mRNA decreased marginally more rapidly than M/L-opsin mRNA, but had also been reduced to 16% of the WT level by P90 (Fig. 6C). Relative to P14, the decrease in M/L-opsin mRNA was not statistically significant at P30 (P=0.09; Fig. 6B), but the level of S-opsin mRNA was significantly lower at the same time point (P<0.05; Fig. 6D). The decreases in both cone opsin mRNAs were statistically significant from P45 onwards. Overall, the qPCR data were concordant with those obtained from immunohistochemistry of wholemounts and transverse sections.
Investigation of dual cones in Rd1 retinas
The second part of this study involved characterizing the population of genuine S-cones, genuine M/L-cones and dual cones in the Rd1 mouse retina at the onset of cone degeneration, P14, and at the approximate midpoint of degeneration, P60. These data are available in the retinas of WT mouse strains [15], but not in the Rd1 retina. Retinal wholemounts were immunolabeled with antibodies to S- and M/L-opsins. Images from the peripheral retina were captured, pre-processed, merged, and the cone densities of genuine S-opsin cones, genuine M/L-opsin cones and dual cones then quantified in each of the four retinal quadrants using colour thresholding (See Methods). At P14, outer segments were quantified, while at P60 cell bodies (plus any surviving outer segments) were used. Initially, however, we tested the methodology using a retinal wholemount from a WT mouse (see Additional files 6 and 7). Examination of this retina revealed 14% genuine S-cones, 35% dual cones and 50% genuine M/L-cones, values that are not dissimilar to those previously reported [15].
At P14, the superior retina (Fig. 7, Additional file 8) comprised a minority of genuine S-cones (15.6%), as expected, with a much higher complement of genuine M/L-opsin cones (38.1%) and dual cones (40.4%). In contrast, the inferior retina (Fig. 7, Additional file 9) contained relatively similar numbers of genuine S-cones (37.5%), genuine M/L-cones (27.2%) and dual cones (35.3%). Nasal (Fig. 7, Additional file 10) and temporal (Fig. 7, Additional file 11) regions had similar numbers of genuine S-cones (41.3%; 41.6%, respectively), genuine M/L-opsin cones (24.2%; 28.5%, respectively) and dual cones (34.5%; 29.9%, respectively).
In agreement with the results from the spatio-temporal characterisation of S- and M/L-cone degeneration, there was a marked divergence in cone composition between the superior (Fig. 8, Additional file 12) and inferior (Fig. 8, Additional file 13) retina at P60. The superior retina at P60 consisted of a majority of dual cones (53.7%), followed by genuine M/L-cones (38.1%) and a sparse population of genuine S-cones (8.2%). In contrast, the inferior retina at P60 contained almost exclusively genuine S-cones (97.7%) with a small minority of dual cones (2.3%), and no identifiable genuine M/L-cones. Nasal (Fig. 8, Additional file 14) and temporal (Fig. 8, Additional file 15) regions had similar compositions of genuine S-cones (23.2%; 30.2%, respectively), genuine M/L-cones (18.6%; 23.7%, respectively) and dual cones (46.3%; 58.1%, respectively). In terms of overall cone survival in the four quadrants at P60, i.e. combined presence of all cone types, the results showed approximately 3-fold greater overall cone survival in the inferior retina (1,154,292 ± 42, 610 pixels2) compared with the superior (342,175 ± 30,593 pixels2), nasal (402,722 ± 59,855 pixels2), and temporal (390,130 ± 52,139 pixels2) quadrants. This results exemplifies the remarkable survival of S-opsin+ cones in the peripheral regions of the inferior retina (see Fig. 8, Additional file 13).
Spatio-temporal characterisation of microglial responses in Rd1 retinas
In the third part of this study we investigated microglial number and activation in the Rd1 retina using well characterised antibodies to Iba1 and CD68. Iba1 is a calcium-binding protein specifically expressed by quiescent and activated microglia, whilst CD68 is a glycoprotein expressed only by activated, phagocytic microglia.
We found the number of Iba1+ and CD68+ microglia in wholemount Rd1 retinas decreased gradually from P21 to P300 (Fig. 9). The majority (79.1%) of microglia at P21 were active and expressed both CD68 and Iba1. These activated microglia presented a typical amoeboid shape with scarce dendrites. By P60, 67% of Iba1+ microglia were still present in the retina and only 17.6% expressed CD68. The inactive microglia displayed typical quiescent morphology with long dendritic processes. By P300, only 33.9% of Iba1+ microglia remained and only 2.6% were active and expressed CD68. Therefore, the activation profile of microglia paralleled the degeneration of S- and M/L-cones in the Rd1 retina, with a high rate of microglial activation occurring at the peak period of cone degeneration.
In transverse sections of the Rd1 retina (Fig. 10), a large population of Iba1+ microglia (62 cells/mm) was present in the ONL and inner/outer segments at P14 where photoreceptor degeneration occurs. These outer retinal microglia were characterised by an activated amoeboid shape, whereas the resident population of Iba+ microglia in the inner retina demonstrated a quiescent (ramified) morphology. The population of Iba1+ microglia in the outer retina was greatly diminished at P21 (26 cells/mm) and decreased gradually thereafter, with very few Iba1+ microglia remaining in the outer retina by P75 (9 cells/mm).