Long-term monitoring of chronic demyelination and remyelination in a rat ischemic stroke model using macromolecular proton fraction mapping

Abstract

Remyelination is a key process enabling post-stroke brain tissue recovery and plasticity. This study aimed to explore the feasibility of demyelination and remyelination monitoring in experimental stroke from the acute to chronic stage using an emerging myelin imaging biomarker, macromolecular proton fraction (MPF). After stroke induction by transient middle cerebral artery occlusion, rats underwent repeated MRI examinations during 85 days after surgery with histological endpoints for the animal subgroups on the 7th, 21st, 56th, and 85th days. MPF maps revealed two sub-regions within the infarct characterized by distinct temporal profiles exhibiting either a persistent decrease by 30%-40% or a transient decrease followed by return to nearly normal values after one month of observation. Myelin histology confirmed that these sub-regions had nearly similar extent of demyelination in the sub-acute phase and then demonstrated either chronic demyelination or remyelination. The remyelination zones also exhibited active axonal regrowth, reconstitution of compact fiber bundles, and proliferation of neuronal and oligodendroglial precursors. The demyelination zones showed more extensive astrogliosis from the 21st day endpoint. Both sub-regions had substantially depleted neuronal population over all endpoints. These results histologically validate MPF mapping as a novel approach for quantitative assessment of myelin damage and repair in ischemic stroke.

Keywords: Macromolecular proton fraction; histology; magnetic resonance imaging; middle cerebral artery occlusion; myelin.

Figure 1.

An example longitudinal observation of the ischemic lesion evolution in the same animal on MPF maps (top row), T₂-weighted images (second row), ADC maps (third row), PD maps (fourth row), and R₁ maps (bottom row) of the rat brain. Images were obtained before, 3 hours after, and then on the 1st, 3rd, 5th, 7th, 14th, 21st, 31st, 42nd, and 56th day after MCAO. Red and green arrows indicate the DE and RE zones within the ischemic lesion, respectively.

Figure 2.

Dynamic profiles of the mean percentage changes in MPF (a), T₂ signal ratio (b), ADC (c), PD ratio (d), and R₁ (e) relative to the pre-surgical baseline assessed over all time points and animals in the DE and RE zones identified within the ischemic lesions (red and green curves, respectively) and their contralateral matching regions (blue and violet curves). Error bars correspond to the standard deviations. Color-coded stars indicate significant differences (*p < 0.05, **p < 0.01, ***p < 0.001) between ipsilateral and contralateral measurements for the DE (red) and RE (green) zones and between the DE and RE zones (black).

Figure 3.

Matched whole-brain MPF maps and histological sections stained with LFB and immunofluoresce-labeled with MBP and GFAP corresponding to the 7th, 21st, 56th, and 85th day endpoints after MCAO. Red and green arrows indicate the DE and RE zones within the ischemic lesions, respectively.

Figure 4.

Magnified views (200× magnification) of the histological sections immunofluorescence-labeled with NF-H (2nd column), MBP (3rd column), co-localized NF-H and MBP (4th column), GFAP (5th column), NeuN (6th column), and co-localized NG2 and DCX (7th column) corresponding to either the DE and RE zones within the ischemic lesion in the animals studied on the 7th (2nd row), 21st (3rd row), 56th (4th row), and 85th (5th row) day after MCAO or similar control locations in a sham-operated animal studied on the 3rd day after surgery (1st row). The locations of magnified regions are labeled in the reference LFB-stained sections (1st column). In all immunofluorescence images, the blue channel depicts the nuclei labeled with DAPI.

Figure 5.

Quantitative histology profiles of the mean percentage changes relative to the contralateral hemisphere in LFB optical density (a), MBP-positive total area (b), NF-positive total area (c), MBP-positive object size (d), NF-positive object size (e), NeuN-positive cell count (f), DCX-positive cell count (g), NG2-positive cell count (h), and GFAP-positive cell count (i) calculated for the DE and RE zones of the ischemic lesion defined according to the MPF maps. The data correspond to the groups of animals studied after sham surgery (n = 3) and on the 7th (n = 2), 21st (n = 1), 56th (n = 2), and 85th (n = 1) day after MCAO. Error bars correspond to the standard deviations. Color-coded stars indicate significant differences (*p < 0.05, **p < 0.01, ***p < 0.001) between ipsilateral and contralateral measurements for the DE (red) and RE (green) zones and between the DE and RE zones (black).