Abstract
Extensive evidence implicates an increase in hippocampal L-type voltage-gated calcium channel (L-VGCC) expression, and ion influx through these channels, in age-related cognitive declines. Here, we ask if this “calcium hypothesis" applies to the neuroretina: Is increased influx via L-VGCCs related to the well-documented but poorly-understood vision declines in healthy aging? In Long-Evans rats we find a significant age-related increase in ion flux through retinal L-VGCCs in vivo (manganese-enhanced MRI (MEMRI)) that are longitudinally linked with progressive vision declines (optokinetic tracking). Importantly, the degree of retinal Mn2+ uptake early in adulthood significantly predicted later visual contrast sensitivity declines. Furthermore, as in the aging hippocampus, retinal expression of a drug-insensitive L-VGCC isoform (α1D) increased – a pattern confirmed in vivo by an age-related decline in sensitivity to L-VGCC blockade. These data highlight mechanistic similarities between retinal and hippocampal aging, and raise the possibility of new treatment targets for minimizing vision loss during healthy aging.
Introduction
Extensive research in the CA1 region of the rat hippocampus has revealed an age-related increase in neuronal Ca2+ influx though L-type voltage-gated calcium channels (L-VGCCs) that is strongly linked with impaired synaptic plasticity and reduced cognitive function [1]–[5]. Diminished visual performance is another important behaviorally-evident functional decline that occurs with aging, beginning in young adulthood, but whose underlying mechanisms are poorly understood [6], [7]. Concurrent declines in neuroretinal function, when measured by electroretinogram (ERG), have also been noted: rod sensitivity and the maximum amplitude of rod responses to light both decrease with age [8], [9]. However, as reviewed by Spear [10], such physiological changes were too modest to account for the age-related vision declines. Here, we test an alternative hypothesis: that changes in retinal ion influx via L-VGCCs occur with age, and are linked to visual performance declines.
To test this hypothesis, we longitudinally and non-invasively measured the extent of retinal ion influx via L-VGCCs in light and dark-adapted retinas using Mn2+-enhanced MRI (MEMRI). In MEMRI, awake and freely-moving animals are injected with a non-toxic dose of the MRI contrast agent Mn2+, a Ca2+ surrogate that accumulates in neurons over a period of a few hours. Later, the animal is anesthetized and extent of retinal Mn2+ uptake is measured using MRI. Importantly, similar to Ca2+, Mn2+ primarily enters neurons through L-VGCCs: In vitro, Mn2+ uptake is strongly inhibited by L-VGCC blockers, and increased both by membrane depolarization (opening L-VGCCs) and the L-VGCC agonist BayK8644 [11], [12]. In vivo studies confirm that Mn2+ uptake is inhibited by the L-VGCC antagonists verapamil [13], nifedipine [14], and diltiazem [15]. Because Mn2+ efflux is slow, taking days to leave the retina [16], uptake measured a few hours after injection is a useful measure of ion influx through L-VGCCs.
In this study, two groups of rats were examined longitudinally – one from young to mid-adulthood, and the other from mid- through old adulthood. MEMRI data were compared to two aspects of visual performance; spatial frequency threshold (‘SFT’; a proxy for visual acuity) and contrast sensitivity (‘CS’). We measured visual performance using optokinetic tracking (OKT), a reflex which requires no animal training and avoids the potential confound of age-related impairments in thermoregulatory function [17]. In both groups, the first and final vision tests were followed by MEMRI: Using an eye patch to keep one eye dark-adapted while the other was exposed to normal lab lighting, we tested for longitudinal changes in retinal ion influx in both lighting conditions. Dark-adapted values represent maximal ion influx in outer retina, which is populated almost exclusively by photoreceptors, while subtracting light from dark values provides quantification of activity-dependent photoreceptor Mn2+ uptake. Anterior to the outer retina resides the inner retina, a brain-like complex of bipolar, amacrine, and ganglion cells that process light information gathered by the photoreceptors. Age-related ion-regulatory changes may also occur in the inner retina. However, the spatial overlap of light-activated and light-inactivated neurons makes unraveling the relationship between activity and Mn2+ uptake in inner retina more difficult than in outer retina. We therefore provide inner retinal data as Supplemental Material, while focusing on the photoreceptor-dominated outer retina to test our hypothesis that changes in retinal ion influx via L-VGCCs occur with age.
In a separate set of experiments, we verified that retinal Mn2+ uptake was inhibited by both topical and systemic L-VGCC blockade. Next, we used Western blots to assess age-related changes in retinal L-VGCC expression, expecting an age-related increase for only the α1D isoform, based on findings from CA1 of the rat hippocampus [18], [19]. Because α1D L-VGCCs are roughly an order of magnitude less-sensitive to antagonism than the α1C isoform [20]–[22], we also used MEMRI to non-invasively test for an age-related loss in sensitivity to L-VGCC blockade.
Results
Visual Performance
We first characterized longitudinal changes in visual performance in Long Evans rats using OKT, which is based on the reflexive head-movements of awake and free-moving rats in the presence of visible stimuli. SFT declined from Young to Mid-adulthood (Group YM; P = 2.4e-4) but remained stable from Mid- to Old adulthood (P>0.26 for all comparisons within Group MO). In contrast, CS declined significantly with age in both Group YM (P = 3.4e-2) and Group MO (P = 2.7e-4 for ∼7 to ∼11.5 mo; P = 1.3e-4 for ∼11.5 to ∼19 mo) (Fig. 1). These results confirm and extend prior findings of visual performance declines with aging in other rodents [23], [24].
Outer Retinal Mn2+ Uptake Changes with Age
Next, we quantified retinal ion influx in vivo using MEMRI. Dramatic age-related increases in retinal Mn2+ uptake were easily visualized against the stable baseline (i.e., no Mn2+) R1 (Fig. 2). Subtracting baseline from Mn2+-enhanced data allowed for quantitative examination of tissue ΔR1s, which are directly proportional to tissue Mn2+ concentration. We found significant age-related increases in outer retinal Mn2+ uptake between young and mid-adulthood (Group YM; dark: P = 3.4e-5; light: P = 1.9e-4), and between mid- and old adulthood (Group MO; dark: P = 1.7e-4; light: P = 3.8e-4) (Fig. 3). These age-related increases in Mn2+ uptake were observed in both light-exposed and dark-adapted (patched) eyes. The inner retina showed similar changes (see Supplemental Figure S3 in File S1).
The expected pattern of activity-dependent outer retinal Mn2+ influx – low in photoreceptors exposed to light, when membranes are hyperpolarized and L-VGCCs are closed, but high in darkness, when photoreceptors are fully depolarized (for review, see Yau, 1994) – was noted at all ages in our longitudinal studies: Dark-light differences in outer retinal Mn2+ uptake were significantly greater than zero (all P<0.018), and dark/light ratios were significantly greater than 1 (all P<5.8e-3) (Fig. 3). The absolute amount of activity-dependent Mn2+ uptake (i.e., dark-light differences in ΔR1) increased significantly with age – both from young to mid- adulthood (Group YM; P = 0.038), and mid- to old adulthood (Group MO; P = 0.027) – but, interestingly, the relative amount (dark/light ratio) did not (P>0.40 in both groups) (Fig. 3). These data suggest that, despite some consistencies in retinal light responses across age groups, there is a robust age-related increase in net ion influx through photoreceptor L-VGCCs.
Outer Retinal Mn2+ Uptake Predicts CS Declines
Having established age-related declines in visual performance together with increases in retinal Mn2+ uptake, we next tested whether high Mn2+ uptake is linked with age-related declines in visual function. Notably, higher-than-average outer retinal Mn2+ uptake on any given rat’s first MRI scan predicted a greater-than-average rate of CS decline in the ∼4.5 mo following the first MRI scan (Fig. 4). This relationship was significant when evaluating patched (dark-adapted) eyes, unpatched (light-exposed) eyes, or activity-dependent (dark-light difference) outer retinal Mn2+ uptake (all r<−0.50; P<3.1e-3). The initial value of CS was also a significant predictor of CS decline, such that rats beginning the study with higher-than-average CS showed the greater-than-average declines in CS (r = −0.51; P = 2.9e-3; Fig. 4). The strongest predictor of CS declines was Mn2+ uptake in the dark-adapted outer retina (P = 1.2e-6; r = −0.74; Fig. 4), and after statistically controlling for that relationship, only initial CS remained a significant predictor of CS decline (P = 3.5e-4; Fig. 4). Importantly, the relationship between initial CS and later CS declines (which may be influenced by ‘regression to the mean’) does not account for the relationship between Mn2+ uptake and CS declines. After statistically controlling for the relationship with initial CS, each of the Mn2+ uptake variables remained a significant (P<1.4e-3) predictor of CS decline, with dark-adapted outer retinal Mn2+ uptake being the strongest remaining predictor (P = 2.1e-7; Fig. 4). We note that regression analyses that used both light-adapted Mn2+ uptake and the dark-light difference as predictors showed that each was a significant and unique predictor of CS declines, either when statistically controlling for the relationship with initial CS (both P<1.8e-4) or not (both P<6.7e-4). In short, both initial Mn2+ uptake and initial CS are strong and unique predictors of future CS declines in the ∼4.5 mo following the first MRI measurement (Fig. 4).
In Group MO, the third measurement of visual performance revealed an additional pattern: From ∼4.5 to ∼12 mo after the first MRI, rats that had already experienced substantial CS declines (from 0 to ∼4.5 mo post-MRI) tended have their remaining function preserved. However, rats with little-to-no CS declines in the first ∼4.5 mo post-MRI experienced substantial declines thereafter. For this reason, high Mn2+ uptake – a strong predictor of CS declines in the ∼4.5 mo following the first MRI – was also a significant predictor of preserved CS in the later period (∼4.5 mo to ∼12 mo post-MRI) (see Supplemental Material (Fig. S4, Table S4) in File S1). Taken together, the initial degree of retinal Mn2+ uptake strongly predicted the timing of an animal’s transition to its old-adult level of visual performance (i.e., an immediate vs. delayed decline).
Outer Retinal Mn2+ Uptake is L-VGCC-Dependent
Having established a robust link between the extent of retinal Mn2+ uptake and future visual declines, we next examined the role of L-VGCCs in photoreceptor (i.e., outer retinal) Mn2+ uptake using the specific L-VGCC blocker nifedipine. Consistent with expectations, nifedipine inhibited outer retinal Mn2+ uptake in dark-adapted (patched) eyes (P = 0.042; Fig. 5), but not in light-exposed eyes (P>0.5). Further analysis revealed that outer retinal dark-light differences were absent in nifedipine-treated rats (P>0.5) but present in vehicle controls (P = 0.018; Fig. 5), such that ratio (ΔR1,dark/ΔR1,light) and difference (ΔR1,dark–ΔR1,light) scores differed significantly between the nifedipine and vehicle groups (P = 0.043 and 0.026, respectively). Analysis with a mixed ANOVA yielded the same result (light vs. dark × drug vs. vehicle interaction: F[1, 12] = 6.5; P = 0.026). In a separate group of rats, we verified that Mn2+ uptake was inhibited by local action of nifedipine, rather than possible systemic effects: In dark-adapted rats, nifedipine eye drops significantly inhibited outer retinal Mn2+ uptake, relative to measurements from the contralateral vehicle-control eyes (paired t-test: P = 0.031; Fig. 5). These data confirm a dominant role of L-VGCCs in outer retinal Mn2+ uptake.
Retinal L-VGCC Expression Changes with Age
Our longitudinal studies demonstrated an age-related increase in retinal Mn2+ uptake, and our experiments with nifedipine demonstrated that a substantial fraction of retinal Mn2+ uptake occurs through L-VGCCs. We therefore tested for an age-related increase in L-VGCC expression. Comparing Western blots from young versus mid-adult rat retinas, we found an age-related increase in expression of the ∼180 kDa α1D isoform (P = 0.043; Fig. 6). Neither the larger (>200 kDa) α1D isoform nor the α1C isoform showed an age-related change in expression (respectively P>0.4 and P>0.2) – similar to the previously-documented pattern at CA1 of the rat hippocampus [18], [19].
We also tested for this age-related, isoform-specific increase in L-VGCC expression in vivo: Because the α1D isoform is roughly an order of magnitude less-sensitive to blockade than α1C [20]–[22], an age-related increase in retinal α1D expression should make it more difficult to block Mn2+ uptake with an L-VGCC antagonist. We measured retinal Mn2+ uptake after injecting low (10–30 mg/kg) or high (100–125 mg/kg) doses of D-cis-diltiazem into dark-adapted young and mid-adult rats. In young adults, both low (10–30 mg/kg) and high (100–125 mg/kg) doses of diltiazem inhibited retinal Mn2+ uptake by ∼40%, relative to age-matched controls (P = 5.5e-3 and 4.7e-3, respectively; Fig. 7). In contrast, the low dose of diltiazem had no effect on retinal Mn2+ uptake in mid-adult rats (P>0.4), but significant inhibition of Mn2+ uptake (P = 1.1e-4) was observed with high doses of diltiazem (Fig. 7). In short, the mid-adult rat retinas appeared less-sensitive to blockade, consistent with Western blot findings of an age-related increase in expression of a drug-insensitive L-VGCC isoform.
Discussion
Previous studies of the rat hippocampus formed the foundation of the ‘calcium hypothesis of aging’ by demonstrating progressive age-related increases in neuronal Ca2+ influx through L-VGCCs, L-VGCC density, and L-VGCC protein expression, which are greatest in those rats with the poorest cognitive function [1]–[5], [18], [19]. In this study, we examined an analogous hypothesis in the neuroretina, and found that age-related declines in CS coincide with increases in retinal ion influx via L-VGCCs. Importantly, the extent of retinal ion influx strongly predicted subsequent rates of CS decline. Furthermore, retinal Mn2+ uptake was regulated by age-related and isoform-specific increases in expression of L-VGCCs, which are widespread in the retina [25], [26] and the largest single pathway for neuroretinal Ca2+ influx [27]–[29]. L-VGCCs are critical to retinal function [30], [31], and acute L-VGCC blockade improves CS in humans (though the potential influence of age has not been investigated) [32], [33]. Long-term changes in retinal L-VGCCs may also alter growth and remodeling of photoreceptor axons [25]. Together, our data unambiguously extend, for the first time, the ‘calcium hypothesis of aging’ to the neuroretina.
We evaluated neuroretinal ion influx through L-VGCCs in vivo with MEMRI, and found the expected pattern of activity-dependent retinal ion influx at all ages (Fig. 3; [16], [34], [35] – low in photoreceptors exposed to light, when membranes are hyperpolarized and L-VGCCs closed, but high in darkness, when photoreceptors are fully depolarized and L-VGCCs open. Mn2+ uptake is robustly sensitive to targeted manipulation of L-VGCCs, as demonstrated by reduced retinal Mn2+ uptake following L-VGCC blockade with nifedipine (Fig. 5) and diltiazem (Fig. 7). That finding is likely due to local drug effects at the retina, rather than systemic (e.g., cardiovascular [36] effects of L-VGCC blockade, since topical and systemic application of nifedipine yielded similar reductions in outer retinal Mn2+ uptake (Fig. 5). We analyzed the role of L-VGCCs on activity-dependent Mn2+ uptake, and found that L-VGCC blockade reduced Mn2+ uptake in the dark-adapted outer retina to light-adapted levels (Fig. 5). We found that the absolute amount of activity-dependent Mn2+ uptake increased with age (Figs. 2, 3). These MEMRI data are in apparent contradiction to results from previous ERG studies in which photoreceptor responses to light decrease with age in humans, mice, and pigmented rats [8], [9], [24]). It seems likely that MEMRI and ERG evaluate different aspects of outer retinal ion channel activity: ERG reports on light-dependent changes in photoreceptor membrane voltage, which are driven by Na+ (and other cation) entry through cyclic guanosine monophosphate-gated channels [37] while MEMRI analyzes photoreceptors’ dark-light differences in ion influx through Ca2+ channels, particularly L-VGCCs. Because ERG declines do not seem to explain visual declines, our present results strongly support MEMRI as a powerful tool for studies into the role of L-VGCCs in aging.
In the rat hippocampus (CA1), expression of L-VGCCs increases with age [1], [18], [19]. To test for this possibility in the retina, we compared Western blots from young and mid-adult adult retinas and found an isoform-specific increase that was strikingly similar to that reported for the rat hippocampus [18], [19]: Expression of the ∼180 kDa α1D isoform increased with age, while expression of α1C and the larger (>200 kDa) α1D isoform appeared independent of age (Fig. 6). Because the α1D isoform is roughly an order of magnitude less-sensitive to pharmacological blockade than α1C [20]–[22], we reasoned that an age-related increase in α1D expression would be revealed by an age-related decrease in drug sensitivity. In vivo, we found substantial inhibition of retinal Mn2+ uptake with 10–30 mg/kg doses of the L-VGCC blocker diltiazem – consistent with previous work [15] – but only in young adult rats. In mid-adult rats, ≥100 mg/kg doses were needed to produce similar levels of inhibition (Fig. 7). Our data strongly suggest that L-VGCC-based therapies/interventions may become progressively less-effective with age because existing drugs are relatively ineffective at the α1D isoform. The sensitivity of MEMRI to age-related changes in L-VGCCs highlights a potential application of this method for in vivo evaluation and optimization of drug efficacy, for instance, complementing ongoing development of L-VGCC blockers specific for the α1D-isoform [38]. Although clinically-available L-VGCC blockers seem to have negligible influence on optical factors like lens accommodation (i.e., ciliary muscle contractility [39] and pupil size [40], [41]), and indeed appear to acutely improve vision in humans [32], [33], preclinical evaluation of new α1D-selective drugs will likely require assessment of the acute and chronic effects on vision. OKT is a promising approach to aid in such assessments, given our present demonstration that OKT is sensitive to age-related and L-VGCC-associated vision declines.
We now consider the following potential confounders to the interpretation of the MEMRI data: (1) Very high doses of Mn2+ carry some risk of toxicity [42], which could negatively impact longitudinal studies. Several lines of evidence argue against Mn2+ toxicity in the present study: We have previously demonstrated that the present dose (222 µmol Mn2+/kg) is non-toxic to the rat retina, based on measurements of intraocular pressure, blood-retinal barrier integrity, retinal histology [34], electroretinography, and dose-redose reproducibility of retinal Mn2+ enhancement [43]. Also, if Mn2+ exposure affected long-term visual function, it would be evident in comparisons of ∼7 mo data from Groups YM and MO: At that time point, the former but not the latter had previously been injected with Mn2+. Post-hoc, we find that Group YM and Group MO have similar visual function at ∼7 mo (for SFT, P = 0.28; for CS, P = 0.091; two-tailed t-tests), further arguing against toxicity. Thus, available data overwhelmingly demonstrate that our present dose of 222 µmol Mn2+/kg is non-toxic to the rat retina. This is consistent with previous work related to the rat hippocampus, which showed no histological signs of toxicity following a 500 µmol Mn2+/kg injection [44], and normal performance on a memory task after a 200 µmol Mn2+/kg injection [45]. (2) Longitudinally, we found that Mn2+ uptake increased with age. If Mn2+ efflux rates were slow enough, then the second measurement might be higher than the first merely because residual Mn2+ remains from the first injection. This seems unlikely, given that the half-life for retinal Mn2+ efflux is less than one day [16]. Furthermore, the longitudinal finding of higher Mn2+ uptake in mid- than young-adult rats (Group YM) can be reexamined by cross-sectional comparison of the once-injected ∼2.5 mo rats from Group YM to the once-injected ∼7 mo rats from Group MO: Those post-hoc comparisons yield significant differences for both inner and outer retina in both dark and light (all P<1.5e-3), consistent with the longitudinal findings (Fig. 3). (3) Finally, any declines in blood-retinal barrier (BRB) integrity might alter neuronal Mn2+ exposure, and therefore uptake. We tested for vitreous enhancement following injection of the intravascular contrast agent Gd-DTPA, and found no signs of BRB compromise during healthy aging (see Supplemental Figure S2 in File S1), consistent with previous work [46].
The present results may explain why prior studies of the anatomy and physiology of the aging retina have had little success explaining age-related declines in visual function (see [10] for review). For example, as detailed in the Supplemental Material (Fig. S3 in File S1), in vivo measurements of retinal morphology did not indicate volume loss with age, suggesting that anatomical measurements aimed at detecting neuron loss would give little insight into vision declines in healthy aging. Also, previous in vivo physiologic measurements using ERG are likely insensitive to the important changes in L-VGCCs found herein, which strongly predicted the rate of age-related declines in CS (Fig. 4). Our demonstration that the ‘calcium hypothesis of aging’ applies to the retina offers a new approach for understanding age-related declines in visual function – one which might be pursued in translational clinical studies using the FDA-approved Mn2+-based contrast agent Teslascan [16].
Materials and Methods
Male Long-Evans rats (Hilltop Labs; Scottdale, PA) were studied. Rats were given food and water ad libitum and housed and maintained in normal 12 h light/12 h dark cycling. Ages, weights, etc. in this section are reported as mean(standard deviation). All animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and authorization by the Institutional Animal and Care Use Committee (IACUC) of Wayne State University.
Animals Studied Longitudinally
Group YM was studied from Young to Mid adulthood, and Group MO was studied from Mid to Old adulthood. At the first and final time points for each group, tests of visual performance were followed (∼1 wk later) by high-resolution MEMRI measurements of both eyes (details below).
The need for high-quality longitudinal MRI data from both eyes of each rat resulted in some missing or excluded data, due, for instance, to anesthetic-related death. When possible, outcome metrics between rats lost to follow-up (missing/excluded) and rats retained for the duration of the study were compared, and as suggested by visual inspection of scatter plots (Figs. 1, 3, and Fig. S3 in File S1), no statistically significant differences were noted.
We started with a total of 42 rats in Group MO. Three rats were excluded from the final analysis because they had unusually and unilaterally thick retinas (|left-right thickness difference| of (mean(SD)) 72(7) µm, versus 9(8) µm in the other rats) to the degree that they were univariate outliers (|z|>3.3). This asymmetry was not related to the eye patch or duration of anesthesia: The thicker retina was in the unpatched eye in one of the three cases, and in the first eye scanned in two of three cases. The remaining 39 rats in Group MO, were 206(10) d old, and weighed 439(50) g at the first time point. In 32 of those rats, high-quality MRI data was collected from both eyes at the first time point. Roughly half were available at the intermediate time point, 144(13) d later, when only visual performance and body weight were measured (n = 19; age 350(15) d; 580(50) g). Fifteen of those rats contributed data to the final (old adult) time point (age 580(29) d; 605(67) g). Of those fifteen, high-quality MRI data could not be collected for one, and another three were randomly selected to be scanned without Mn2+ injection, so as to measure baseline retinal R1 in old adulthood. In summary, paired (longitudinal; mid- vs. old adult) comparisons of visual performance were performed in 15 Group MO rats, 14 of which were used for comparisons of eye morphology, and 11 for comparisons of retinal Mn2+ uptake.
We started with 22 rats in Group YM. One rat had unilaterally thickened retina and was excluded from all analyses. Of the remaining 21 rats (initial age of 73(9) d; weighing 290(70) g), 18 provided high-quality MRI data at the first time point. Thirteen of those were available for follow-up in mid-adulthood (aged 227(7) d; 502(34) g), of which 11 provided high-quality MRI data. In summary, paired (longitudinal; young vs. mid-adult) comparisons of visual performance were performed in 13 Group YM rats, 11 of which were used for comparisons of both eye morphology and retinal Mn2+ uptake.
Visual Performance
Visual performance was measured in awake and free-moving animals using OKT, which utilizes rodents’ reflexive head movements in the presence of a moving sine wave grating. Head movements are present when stimuli are clearly visible, but absent when the experimenter makes stimuli too difficult to see, either due to low contrast when measuring CS, or high spatial frequency when measuring SFT. Further details on OKT, including video-recorded examples of the characteristic head movements and experiments on the neuronal pathways involved in OKT responses, are available through the work of Douglas, Prusky, and colleagues (e.g. [47], [48]). In the present studies, SFT and CS were measured on an in-house built OKT device (Fig. 1, File S1 in File S1). Rats had unrestricted head movement during each of the eight 15–20 min sessions (1–2 per day) used to measure SFT (4 of the 8 sessions) and CS at a given age. The best performance recorded for an animal during these sessions (confirmed by multiple tracking events at the same OKT device setting) was used for statistical comparisons.
Eye Patch
An opaque eye patch was used to keep one eye dark-adapted while the other was exposed to normal lab lighting (∼300 l×) during Mn2+ uptake. The eye to be patched was alternated from rat to rat, so that roughly half the members of each group had the left eye patched at each time point. The day before MRI scans, the patch was adhered to one side of the head while the rat was anesthetized with diethyl ether, as previously described [35]. Briefly, after the selected eye was gently sutured shut and protected by application of puralube (Pharmaderm; Melville, New York), the patch was adhered with a combination of eyelash glue (Andrea modlash adhesive; American International Industries, Los Angeles, CA) and spirit gum (Mehron Inc., Chestnut Ridge, NY). Each rat was fit with an Elizabethan collar and monitored until recovery from anesthesia (≤15 min). Rats were then dark-adapted overnight with free access to food and water. In all cases, sutures remained intact and patches remained fully-adhered until MRI scans on the following day.
Acquisition of MEMRI Data
Prior to i.p. injection of 44 mg MnCl2·4H2O/kg body weight (0.1 M solution in 0.9% saline), rats were brought into normal lab lighting for 30 min. After Mn2+ injection, rats continued their monocular exposure to normal lab lighting for ∼4 h – our standard post-injection time span, selected to ensure full retinal Mn2+ uptake while animals remained awake and freely moving [14]–[16], [34]. MRI readout of retinal Mn2+ uptake was performed under dim red light or darkness, and began immediately after that ∼4 h period, when rats were anesthetized with a ketamine/xylazine (‘k/x’) solution. Maintenance doses of k/x were administered in situ as needed through an intraperitoneal line accessed from just outside the magnet bore.
MRI scans of the left eye began 4.4(0.4) h after Mn2+ injection, and were immediately followed by scans of the right eye (5.5(0.7) h post-Mn2+). For each eye, tissue R1 ( = 1/T1) was measured as follows: Using a 1.0 cm diameter receive-only coil on a 7 T Bruker ClinScan system, retinal images were collected at eight different TRs with a standard spin-echo sequence (echo time (TE) 13, 160×320 matrix, slice thickness 600 µm; 8×8 mm2 field of view; yielding an in-plane resolution of 50 µm from superior to inferior×25 µm in the axial (optic nerve to cornea) direction). Multiple repeat images were collected at lower TRs (total number given in brackets), then registered and averaged offline to improve signal-to-noise. Images were collected in the following order: TR 0.15 s [6], 3.50 s [1], 1.00 s [2], 1.90 s [1], 0.35 s [4], 2.70 s [1], 0.25 s [5] 0.50 s [3].
Acquisition of Baseline (no Mn2+) Data
To aid in the interpretation of MEMRI data, the above procedures for measuring R1 in a light-exposed and dark-adapted eye of the same animal were performed in 9 rats without Mn2+ injection. The patched and unpatched eyes of young (n = 3, aged 74(5) d, weighing 296(17) g), mid- (n = 3, 198(7) d, 417(25) g) and old adult rats (those randomly selected from Group MO; n = 3, 570(29) d, 557(60) g) were scanned under k/x anesthesia, starting 5.1(0.6) h after exposure to normal lab lighting began. This timing closely matched that used for Mn2+-injected rats. Because baseline R1s were stable with age and light exposure (Fig. 2), we averaged across ages and lighting conditions to generate a single baseline outer retinal R1 (0.568 s−1), which was subtracted from Mn2+-enhanced R1s to calculate ΔR1s. In a subset of these baseline rats (2 young, 1 mid, 2 old), R1 measurements were followed by evaluation of blood-retinal barrier (BRB) integrity using dynamic contrast enhanced MRI as detailed in Supplemental Material (Fig. S2 in File S1).
Spatial Normalization of Retinal Data and Analyses of Morphology
Previous rat (and human) studies have demonstrated decreases in retinal thickness and increases in retinal surface area throughout adulthood (e.g., [49]–[52]). Spatial normalization of retinal data is therefore an important precursor to physiological (i.e., Mn2+ uptake) comparisons, and was performed as detailed by Bissig & Berkowitz [35] using semi-automated R (http://www.r-project.org) scripts developed in-house. Briefly, polynomials were fit to the to the vitreoretinal border, then integrated about the central axis of the eye to calculate retinal surface area and (in combination with retinal thicknesses; see below), retinal volume. In-plane, signal intensities were sampled along perpendiculars to the polynomials, then organized as a linearized image of the retina. The distance from optic nerve to ciliary body was measured for each hemiretina, and values were averaged to calculate retinal extent. The linearized retina was then binned in 10% increments of that distance, % extent, with 0% extent at the optic nerve head, and 100% extent at the ora serrata. Average signal intensity as a function of retinal depth was calculated for each % extent bin, producing a signal intensity profile. Vitreoretinal and retina/choroid borders were demarked in each profile (where signal intensity fell halfway between the local minimum and maximum) and subtracted to calculate retinal thickness. Profiles were then resampled from a µm scale to a % thick scale, with 0% thick at the vitreoretinal border, and 100% thick at the retina/choroid border, in 4% thick increments. These spatially-normalized signal intensity profiles facilitated comparisons of retinal Mn2+ uptake: Although the retina thins with age and distance from the optic nerve, the relative (% thick) position of each retinal layer is stable in healthy adults [49], [53]. We report average retinal thicknesses and tissue Mn2+ uptake for the central retina (10–30% extent).
Retinal morphology, produced as a byproduct of spatial normalization and not the focus of the present work, is summarized in the Supplemental Material (Fig. S3 in File S1). There, we also provide measurements of eye size, optical components, and the resulting estimates of refractive state [54]. We note that regression analyses revealed no consistent relationships between eye/retinal morphology and retinal Mn2+ uptake (Supplemental Tables S1, S3, and S4 in File S1).
Analysis of Retinal Mn2+ Uptake
As described above, central retinal signal intensity (‘SI’) profiles were measured at each TR. These signal intensities were used to calculate tissue T1 based on the equation SI = a+b * (exp(-TR/T1)), where a, b, and T1 are fitted parameters. Data were fit with this function by the Levenberg-Marquardt nonlinear least-squares algorithm using the minpack.lm library for R. Binned central retinal data were then averaged to produce a single profile of T1 as a function of depth into the retina (i.e., % thick). T1s from 16–28% thick were averaged to represent inner retina, while data from 48–68% thick were averaged to represent outer retina. These spans respectively fall within the inner plexiform and outer nuclear layers, based on in vivo OCT images of Long-Evans rat retinas [55]–[57]. The inverse of T1 is R1, which varies linearly with tissue Mn2+ concentration [58]. Mn2+ uptake was measured by calculating the difference between Mn2+-enhanced R1 and the baseline (i.e., without Mn2+ injection) retinal R1, yielding the measurement ΔR1. Based on measurements from the rat brain [58], ΔR1s of 0.0, 0.5, and 1.0 s−1 represent tissue Mn2+ concentrations of 0, 80, and 160 µM respectively.
Statistics for Longitudinal Studies
We began by evaluating baseline (i.e., no Mn2+) R1s. Since these data were collected in young, mid-, and old-adult rats, we first tested (linear regression) for effects of age by looking for significant correlations between age and either R1,Dark, R1,Light, or the dark-light difference (R1,Dark–R1,Light). These analyses suggested, as expected, no effect of age on baseline values (see legends for Fig. 2 as well as Fig. S3 in File S1). Since we also found no effect of light on baseline R1s (two-tailed t-tests; n = 9 pairs of eyes), we averaged across ages and lighting conditions to generate a single baseline value for inner retinal R1 (0.514 s−1) and outer retinal R1 (0.568 s−1). These R1s were subtracted from the Mn2+-enhanced R1s, discussed next, to generate ΔR1s.
Based on previous in vivo MEMRI studies (e.g., [34], [35]) demonstrating that photoreceptor ion influx is greatest in darkness when photoreceptors there are fully depolarized [59], outer retinal Mn2+ uptake is expected to be higher in the patched, dark-adapted eye than in the unpatched, light-exposed eye. Here, we performed one-tailed paired t-tests to test for that pattern. We also generated ΔR1,Dark/ΔR1,Light ratios, and tested whether they differed significantly from 1 (one-sample t-tests).
Next, we tested for longitudinal changes in each variable. Paired two-tailed t-tests were used to compare the first to final time point within each group, testing, for instance, whether retinal Mn2+ uptake increased significantly between ages ∼2.5 and ∼7 mo in Group YM. To control for type I error in these multiple comparisons, only results falling below a standard false discovery rate threshold (‘FDR’) were considered significant (q = 0.05 on 52 tests (including comparisons of retinal morphology, etc.; see Fig. S3 in File S1); [60]).
Linear regression was used to test whether the initial value of a given variable predicted the rate of change in a given variable. For instance, do rats with above-average Mn2+ uptake in young-adulthood experience above-average declines in CS in the subsequent 4.5 mo? For a given time span and variable (‘VAR’), rates were calculated as (VARfinal – VARinitial)/(ln(agefinal) – ln(ageinitial)). For Group MO, average rates of change from study start to study end in SFT, CS, and body weight – variables which were also measured at an intermediate time point ∼4.5 mo after the initial MRI – were treated in the same way, but using a linear best fit (after log-transforming age) to the three available measurements.
Additional regression analyses were used, for instance, to test whether two variables are correlated with one-another at the first time point. They are detailed as Supplemental Material (Tables S1–S4 in File S1).
Where possible, regression analyses are performed after combining data from Groups YM and MO: Values from each group at each age – and for rates at each time span – are standardized by conversion to z-scores: For instance, a Group YM subject’s standardized retinal thickness at age ∼2.5 mo is ([subject’s retinal thickness]-[mean of YM retinal thicknesses at ∼2.5 mo])/[SD of YM retinal thicknesses at ∼2.5 mo]. Standardizing scores has no effect on p-values or correlation coefficients (Pearson’s r) when testing one group at a time, but allows both groups to be combined in the same analysis without biasing the outcome due to differences in group means or variances. Before finalizing comparisons, we also tested for Group × variable interactions. These could occur if, for instance, Mn2+ uptake was related to CS in mid-, but not young, adults. In the presence of a suspected interaction (P<0.05; not corrected for multiple comparisons) groups were analyzed separately. When there was no evidence of an interaction (P>0.05) formal statistical testing used only the combined (YM and MO) analysis. For completeness, though, we report correlation coefficients from each group.
Regression results were considered significant below a standard FDR threshold (q = 0.05; see Tables S1–S4 in File S1 for total number of tests). Some post-hoc testing with multiple regression was used to further interpret positive results, and exact p-values are reported in those cases.
MRI Experiments with L-VGCC Blockers
Unless otherwise noted, all aspects of these experiments – for instance, the eye patch procedure, Mn2+ doses, MRI procedures and image processing, including the use of the averaged baseline (no Mn2+) data described above to calculate ΔR1s – are identical to those used in our longitudinal studies. L-VGCC blockers were purchased from Sigma-Aldrich (St. Louis, MO).
Experiment with i.p. nifedipine
Retinal Mn2+ uptake was measured in both light- and dark-adapted eyes using two groups of young-adult rats. After a patch was applied to one eye of each rat (for four members of each group, the right eye), animals were dark-adapted overnight. Immediately after beginning monocular light (∼300 l×) exposure on the following day, drug-treated rats (n = 8; 264(20) g; aged 63(3) d) were injected with nifedipine (i.p., 30 mg/kg) dissolved in DMSO (20 mg nifedipine/ml of undiluted dimethyl sulfoxide), and vehicle-control rats (n = 6; 259(57) g; aged 63(9) d) were injected with DMSO only (1.5 ml/kg). Each rat was injected with Mn2+31(2) min later, then maintained in normal lab lighting until the start of anesthesia (k/x) and immediate MRI scanning of the left, then right, eyes (respectively 4.3(0.2) and 5.5(0.2) h after Mn2+ injection in drug-injected and 4.4(0.3) and 5.6(0.3) h in controls).
Experiment with topical nifedipine
The influence of nifedipine eye drops on retinal Mn2+ influx was tested in five dark-adapted young adults (330(18) g; aged 80(2) d). Undiluted PEG400 was used as a vehicle for the extremely hydrophobic nifedipine. All procedures took place under dim red light or darkness. After anesthetizing the rat with diethyl ether, six 50 µl drops of nifedipine solution (0.211 M) were applied to one eye (the right in 3 of 5 subjects; ∼1 min between each drop), and six 50 µl drops of vehicle (PEG400 only) were applied to the contralateral eye – allowing for paired comparison of nifedipine-exposed to control eyes. No signs of irritation were noted when following application of PEG400 and nifedipine, consistent with previous work [40], [61]. Rats were injected with Mn2+38(2) minutes after nifedipine exposure (ensuring full recovery from anesthesia), then scanned ∼4 h later (left and right eye respectively at 4.0(0.3) and 5.3(0.5) h post-Mn2+). Scans began immediately after inducing urethane anesthesia (3.7(0.3) ml/kg of a 36% w/v solution in 0.9% saline). Only dark-adapted retinas were studied in this experiment; eye patches were not applied to these rats.
Experiment with i.p. diltiazem
We used D-cis-diltiazem (i.e. the (+)-cis isomer, which has high affinity and specificity for L-VGCCs [62], [63] for this dose-response experiment based on our prior experience with this drug in young adult rats [15]. Young adult (n = 7, aged 72(2) d, weighing 287(26) g) and mid-adult (n = 10, 202(20) d, 465(59) g) rats were dark-adapted overnight. The following day, they were injected with 10, 30, 100, or 125 mg diltiazem (in 0.9% saline; 1.4 ml/kg body weight), and maintained in darkness until MRI scanning was complete. Rats were injected with Mn2+31(2) min after diltiazem injection. 4.0(0.3) h after Mn2+ injection, the left eye of these dark-adapted rats was scanned under urethane anesthesia (see above; 3.6(0.8) ml/kg). We used age-matched dark-adapted (patched) data from our longitudinal studies as a no-drug control: Young-adult control data was provided by the first time point in Group YM, while mid-adult control data came from the second time point in Group YM and first time point in Group MO.
Statistics
Multiple in vitro [11], [12] and in vivo [13], [15] studies have demonstrated that L-VGCC blockade inhibits Mn2+ uptake. In addition, several previous studies, both in vivo with MEMRI [16], [34], [35] and ex vivo (see [59] for review), have demonstrated that the outer retina is more ion-permeable in dark than light. For these experiments with L-VGCC blockers, it was therefore appropriate to analyze Mn2+ uptake (ΔR1) with one-tailed t-tests (patched>unpatched; vehicle-control>nifedipine-exposed; control>low-dose diltiazem>high-dose diltiazem; α = 0.05). The raw data (scatter-plots) and exact p-values are provided for these tests. We emphasize that the effect of L-VGCC blockade on Mn2+ uptake was highly reproducible across drugs and delivery methods, and the dark-light difference found in controls (for the i.p. nifedipine group) matched findings from our longitudinal studies. Other statistical tests, used sparingly to aid in the interpretation of results, are detailed in Results or Supplemental Material sections.
Western Blots
Retinal expression of two isoforms of the pore-forming subunit of L-VGCCs – α1C and α1D (i.e.Cav1.2 and Cav1.3, respectively) – was measured in young adult (n = 6; all age 59 d, weighing 267(9) g) and mid-adult rats (n = 6; 192 d; 532(53) g). Immediately after death via urethane overdose, the left retina was isolated from each rat and stored at −80°C. Later, samples were sonicated on ice in a nonionic denaturing urea buffer (6 M urea; 62.5 mM Tris-HCl; pH 6.8; 10% glycerol; 2% sodium dodecyl sulfate; 0.00125% bromophenol blue; and freshly-added 5% β-mercaptoethanol; [64], assayed for protein concentration [65] and diluted with the urea buffer to 3 µg protein/µl in preparation for SDS-PAGE. Samples (60 µg protein per lane) and size standards (Bio-Rad Dual Color Standards #161-0374) were loaded onto 6% polyacrylamide gels, then separated and electrotransfered onto PVDF membrane (Immobilon-P, Millipore Corp., Bedford MA). Membranes were blocked at room temperature in TST (10 mM Tris base and 145 mM NaCl in dH2O, pH 8.0, mixed with Tween 20 (0.05% v/v)) containing 10% w/v non-fat dry milk and 3% w/v BSA, then incubated overnight at 4°C with primary antibodies – mouse anti-β actin (clone AC-15, #A5441, lot 030M4788, 1∶10,000 dilution, Sigma-Aldrich) and either mouse anti-α1C (clone L57/46, #73-053, lot 437-4VA-62, 1∶4 dilution) or mouse anti-α1D (clone N38/8, #73-080, lot 437-4VA-10, 1∶4 dilution) obtained from the UC Davis/NIH NeuroMab Facility – mixed into TST containing 5% w/v nonfat dry milk and 1.5% w/v BSA. The following day, blots were thoroughly washed in TST, then incubated for 1.5 h at room temperature with horseradish peroxidase-linked sheep-anti-mouse IgG (#NA931V, lot 399402, 1∶5000 dilution, GE Healthcare), mixed into TST containing 5% w/v non-fat dry milk and 1.5% w/v BSA. Blots were visualized using chemiluminescent horseradish peroxidase substrate (#WBKLS0500, Millipore) and digitally captured using Fluor-Chem E camera system (Protein-Simple Co., Santa Clara CA).
Blot chemiluminescence intensities were quantified in ImageJ. Although we initially considered using β-actin for normalization, this was not practicable due to overly intense β-actin signal [66], which we attribute to the relatively large sample loadings needed to quantify expression of the less-abundant L-VGCCs. Intensities were therefore normalized to a non-specific band common to both anti-α1C- and anti-α1D-exposed blots at ∼60 kDa, as in [67].
Statistical analysis of western blots
The direction of findings for the ∼180 kDa isofrom of α1D was predicted both by two previous studies in CA1 of the rat hippocampus [18], [19], and significant differences in diltiazem dose-response data (see Results). We therefore used one-tailed t-tests (mid>young adults, α = 0.05) to test for age differences in expression of that protein. The other bands (α1C, and α1D banding at >200 kDa) were similarly tested, but served as negative controls: Only the ∼180 kDa α1D isofrom was expected to change with age [18], [19].
Supporting Information
Funding Statement
This study was supported by National Institutes of Health (NIH) EY018109 (BAB), Juvenile Diabetes Research Foundation (BAB), NIH AG034752 (DB), Wayne State University School of Medicine MD/PhD program (DB), and an unrestricted grant from Research to Prevent Blindness (Kresge Eye Institute). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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