Manganese-enhanced MRI studies of alterations of intraretinal ion demand in models of ocular injury

Abstract

Purpose: To provide proof-of-concept that the extent of intraretinal manganese uptake after systemic MnCl(2) injection, detected with manganese-enhanced MRI (MEMRI), assesses alterations in intraretinal ion demand in models of ocular insult.

Methods: In Sprague-Dawley rats, retinal ion demand and thickness were measured from MEMRI data collected before, 4 hours after, or 1, 3, and 7 days after intraperitoneal injection of MnCl(2). Choroidal contribution or blood-retinal barrier permeability surface area product (BRB PS') was determined using MRI after Gd-DTPA injection. Ocular injury was evaluated 24 hours after intravitreal injection of phosphate-buffered saline (PBS, vehicle) or PBS + ouabain, or after intraperitoneal injection of sodium iodate. Manganese retinal toxicity was assessed by comparing full-field, white-flash electroretinographic (ERG) data obtained before and after systemic MnCl(2) administration. Rat choroidal thickness was measured from cross-sections prepared from paraformaldehyde-perfused adult rats.

Results: Comparing pre- and post-Gd-DTPA images demonstrated minimal choroidal contribution to intraretinal analysis. Intraretinal signal intensity returned to baseline by 7 days after MnCl(2) injection. After ouabain injection, receptor and postreceptor uptake of manganese were subnormal (P < 0.05). After sodium iodate exposure, intraretinal manganese uptake was supernormal (P < 0.05) and did not increase with increasing BRB PS'. ERG data did not show any effect of MnCl(2) on photoreceptor a-wave and postreceptor b-wave relative to baseline at either observation time.

Conclusions: MEMRI measurements of uptake of systemically administered and nontoxic doses of manganese appear to be a powerful approach for measuring alteration in intraretinal ion demand in models of ocular injury.

Figure 1

Top: representative high-resolution MRI image of rat eye 4 hours after intraperitoneal MnCl₂ injection. Small white arrows: region in superior retina from which linearized pseudocolor images (bottom) are derived. Bottom: representative regions-of-interest from the same rat before Gd-DTPA (left), after Gd-DTPA (middle), and the difference (right). The same pseudocolor scale was used for all three linearized images, where blue to green to yellow to red represent lowest to highest signal intensity. Uupper dotted white line: boundary between postreceptor and receptor retina of the control retinas demonstrated in previous studies., Bottom dotted white line: boundary of the posterior aspect of the control retinas. These data suggest a minimal choroidal contribution to intraretinal analysis.

Figure 2

Histogram of measurements of corrected central choroidal thickness from cross-sections through four eyes of Brown Norway rats transcardially perfused with 4% paraformaldehyde. Values are increased by 25% to correct for tissue shrinkage during fixation. The distribution is not uniform because the choroid becomes thinner as peripheral eccentricities are approached.

Figure 3

MEMRI signal intensity changes in receptor (Rec.) and postreceptor (Post-Rec.) retina PBS-injected eyes (PBS) and ouabain-injected eyes (OUA) measured 4 hours after intraperitoneal systemic administration of MnCl₂ from the region defined in Figure 1. Solid bars: dark-adapted groups. Significant differences between PBS-injected eyes and ouabain-injected eyes (*P < 0.05) were found. Note that the minimum value of the y-axis is set to the signal intensity measured in the absence of manganese exposure (i.e., 55). Error bars represent SEM, and numbers above the bars give the number of animals studied.

Figure 4

(A) MEMRI signal intensity changes in receptor (rec.) and postreceptor (post-rec.) retina in control (C) and sodium iodate (SI)–treated rats measured 4 hours after intraperitoneal systemic administration of MnCl₂ from the regions defined in Figure 1. Open bars: light-adapted groups. Significant differences (*P < 0.05) were noted. Note that the minimum value of the y-axis is set to signal intensity measured in the absence of manganese exposure (i.e., 55). Error bars represent SEM, and numbers above the bars give the number of animals studied. (B) Plot of BRB PS′versus MEMRI signal intensity changes in receptor (■) and postreceptor (●) for each rat. A significant negative correlation between BRB PS′ and MEMRI signal was found only for the receptor layer (r² = 0.78; P < 0.05) and not the postreceptor layer (r² = 0.25; P > 0.05). The lack of correlation in the postreceptor retina may be attributed to its lower signal-to-noise ratio than in the receptor layer. In any event, after sodium iodate exposure, retinal manganese uptake did not increase with increasing BRB PS′.

Figure 5

Effect of MnCl₂ injection on retinal function. (A) Signals collected 1 week before (thin traces) and 4 hours after (thick traces) drug treatment. Stimulus exposure is given on the left. (B) The leading edge of the photoreceptor response (box, A) is modeled according to a delayed Gaussian model (traces) of phototransduction (P3) to give phototransduction amplitude (Rm_P3) and sensitivity (S). (C) Average (± SEM; n = 6) Rm_P3 1 week before (filled), 4 hours after (gray, n = 6), and 7 days after (unfilled, n = 6) intraperitoneal injection of MnCl₂. (D) Average (± SEM) phototransduction sensitivity for the same time periods.

Figure 6

Effect of MnCl₂ injection on the postreceptor (P2) intensity response function. (A) The amplitude of the inner retinal P2 (raw – P3) was assessed by extracting the amplitude at a fixed criterion time of 110 ms. Average (± SEM) data are shown for signals collected at baseline (unfilled, n = 6) and 4 hours after injection (filled). Intensity response functions are described using a Naka-Rushton function to give saturated amplitude (V_max), semisaturation (K), and slope (n). (B) Average (± SEM) V_max 1 week before (filled) and 4 hours (gray) and 7 days (unfilled) after IP injection of MnCl₂. (C) Average (± SEM) semisaturation. (D) Average (± SEM) slope.

Figure 7

(A) Time course of mean manganese enhancement and clearance in receptor (●) and postreceptor (▲) retina before (time = 0) and after a single IP MnCl₂ injection in dark-adapted rats. Solid lines: best-fit curves to the data using a three-parameter exponential decay model. Open symbols: average data from the same group of rats (n = 4). Mean data from two other groups of rats are also presented: baseline (n = 4) and days 1 and 3 (n = 3) after manganese injection. (B) Reproducibility of receptor (Rec.) and postreceptor (Post-Rec.) manganese enhancement in same group of rats indicated by the open symbols in (A) but given a second MnCl₂ injection 2 weeks after the first injection (retest). Error bars represent SEM, and numbers above the bars give the number of animals studied.