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Comparative Study
. 2014 Apr;27(4):390-8.
doi: 10.1002/nbm.3073. Epub 2014 Jan 16.

Manganese-enhanced MRI (MEMRI) via Topical Loading of Mn(2+) Significantly Impairs Mouse Visual Acuity: A Comparison With Intravitreal Injection

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Free PMC article
Comparative Study

Manganese-enhanced MRI (MEMRI) via Topical Loading of Mn(2+) Significantly Impairs Mouse Visual Acuity: A Comparison With Intravitreal Injection

Tsen-Hsuan Lin et al. NMR Biomed. .
Free PMC article

Abstract

Manganese-enhanced MRI (MEMRI) with topical loading of MnCl2 provides optic nerve enhancement comparable to that seen by intravitreal injection. However, the impact of this novel and non-invasive Mn(2+) loading method on visual function requires further assessments. The objective of this study is to determine the optimal topical Mn(2+) loading dosage for MEMRI and to assess visual function after MnCl2 loading. Intravitreal administration was performed to compare the two approaches of MnCl2 loading. Twenty-four hours after topical loading of 0, 0.5, 0.75, and 1 M MnCl2 , T1 -weighted, T2-weighted, diffusion tensor imaging and visual acuity (VA) assessments were performed to determine the best topical loading dosage for MEMRI measurements and to assess the integrity of retinas and optic nerves. Mice were perfusion fixed immediately after in vivo experiments for hematoxylin and eosin and immunohistochemistry staining. Topical loading of 1 M MnCl2 damaged the retinal photoreceptor layer with no detectable damage to retina ganglion cell layers or prechiasmatic optic nerves. For the topical loading, 0.75 M MnCl2 was required to see sufficient enhancement of the optic nerve. At this concentration the visual function was significantly affected, followed by a slow recovery. Intravitreal injection (0.25 μL of 0.2 M MnCl2 ) slightly affected VA, with full recovery a day later. To conclude, intravitreal MnCl2 injection provides more reproducible results with less adverse side-effects than topical loading.

Keywords: Animal model study; Diffusion tensor imaging (DTI); Diffusion Methods; Exogenous Contrast Methods; Manganese enhanced MRI (MEMRI); topical loading; intravotreal injection; visual acuity.

Figures

Figure 1
Figure 1
Representative T1W images with 1M topical MnCl2 loading acquired from a mouse brain at 24 hours after loading (A), ipsilateral retina and optic nerve (ON), and contralateral lateral geniculate nucleus (LGN) and superior colliculus (SC) are enhanced with topical loading (arrows). The oblique images (B) bisecting retina and optic nerve after topical loading of 1, 0.75, and 0.5M MnCl2 suggest that 0.75M MnCl2 was sufficient to produce discernible enhancement of optic nerve. Bar graphs (C, mean ± SD) demonstrate the signal intensity ratio between labeled and control retina, ON, and SC. With 1 and 0.75M MnCl2 loading, a significant enhancement of the structures along the visual system (retina, ON, LGN, SC) was clearly seen. In contrast, only slight retinal enhancement was seen with 0.5M MnCl2 loading. Therefore, 0.75M was the concentration selected for topical loading in this study. * indicates that P < 0.005 compared to saline group via Tukey HSD test.
Figure 2
Figure 2
Visual acuity was measured daily in eyes after topical loading with 0.75 and 1M MnCl2 (open symbols), and in contralateral, control eyes (filled symbols). Tests for difference between the dose groups indicate that there is no difference at baseline or on the first day of treatment. Between days 2 and 7, however, the difference between loaded and control eyes is not the same in the two different dosage groups (p-values < 0.0001 at days 2–7). At one day after 1M of MnCl2 loading, the visual acuity decreased to zero (completely blind) and did not recover throughout the 7-day time course. In contrast, one day after 0.75M of MnCl2 loading visual acuity decreased to 0.1 c/d and recovered beginning on Day 2 (p < 0.05, compared to 1M loading eyes) until recovering back to the normal range by Day 6. Note 1: mean VA of normal B6 mice (n=30) is 0.36 ± 0.03 (c/d; mean ± SD) Note 2: c/d denotes units of cycles per degree
Figure 3
Figure 3
Brain DTI maps, containing prechiasmatic optic nerves, from mice at one and seven days after topical loading of 1M MnCl2 were examined to assess the effect of Mn2+ on axonal integrity. The region-of-interest (ROI) based analysis was performed on diffusion metrics, including fractional anisotropy (FA), axial (λ||), and radial (λ) diffusivity, from each optic nerve (A, outline in red). At one and seven days after 1M of MnCl2 loading, there was no difference in λ|| and λ between the optic nerves from the control (B, black bars) and loaded (white bars) eyes suggesting no axon or myelin injury upon Mn2+ loading. The matched immunohistochemical staining of phosphorylated neurofilament (SMI-31) and myelin basic protein (MBP) strongly support the axonal and myelin sheath integrity seen by in-vivo DTI (C).
Figure 4
Figure 4
In-vivo T2-weighted (T2W) imaging was performed to compare the retinal thickness between the saline sham) (A) and MnCl2-loaded (B) eyes. Zoom-in images of mouse retina (C and D) seven days after topical loading of 1M of MnCl2 exhibit typical T2W MRI features of a mouse retina wherein sclera appears dark (white stars), choroid is bright (black arrows), and retinal layers (white double-headed arrows) exhibit intensities between sclera and choroid. Clear thinning in Mn2+ loaded retina was seen 7 days after topical loading in T2W MRI. The hematoxylin and eosin (H&E) staining of the frozen-embedded retina confirmed the MRI detected retinal thinning (E). At 24 hours after 1 M MnCl2 loading, heterogeneous retinal swelling was observed. On day 7 after 1M MnCl2 loading, photoreceptor layers degeneration was visible. 0.75 M MnCl2 loading caused retina slightly swelling at day 1 but recovery at day 5 (E). To quantify the extent of retinal thickness change, H&E images were analyzed (F). A linear model was used to estimate mean retinal thickness on a log scale in each of the 5 study groups. Each of the MnCl2 treated groups was compared with the saline control. P-values for the difference in mean retinal thickness are adjusted for multiple testing using Tukey’s HSD test. One day after 1M MnCl2 loading, transient significant swelling of retina (p < 0.005, with blindness) was seen followed by significant retinal thinning at Day 7 (p < 0.005, with no recovery of visual function). At 0.75M of MnCl2 loading, mild retinal swelling was seen at 1 day (significantly-decreased VA without blindness), which returned to normal thickness 7 days after loading (visual acuity returned to normal by 5–6 days post-loading). Diamond symbols: mean value
Figure 5
Figure 5
The effect of intravitreal Mn2+ injection on retinal integrity and visual function was examined on mouse eyes receiving 0.25 and 2 _L saline injection. The effect of injecting 0.25 and 2 _L on the visual acuity (VA) was examined where 2_L saline injection transiently impaired visual function at 4 hours and Day1 after injection, subsequently recovered (VA ≥ 0.3) at Day 2 (A). The injection of 0.25_L of saline did not impact VA (A). Representative T1W images (B) obtained at 24 hours post-injection with the working dosage (0.25_L and 0.2M MnCl2, 50 nmol) demonstrate the effect of Mn2+− induced intensity enhancement in the loaded visual pathway (left retina, left optic nerve, right LGN, and right SC). The VA was affected by the working dosage at 4 hours post-injection with nearly full recovery by 24 hours post-injection (A). Retinal thickness was examined with H&E staining of frozen-embedded mouse eyes (C and D). The previously reported safe intravitreal 2 _L saline injection caused retinal swelling at Day 2. In contrast, the other reported safe intravitreal saline injection (0.25 _L) indeed did not cause any retinal swelling of the injected eyes (C and D). MnCl2 injection caused slight retinal swelling at 4 hours that recovered at 24 hours post-injection. Histologically-revealed retinal thickening paralleled the decreased VA caused by injection volume and Mn2+ (A). Diamond symbols: mean value

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