Inflammation Combined with Ischemia Produces Myelin Injury and Plaque-Like Aggregates of Myelin, Amyloid-β and AβPP in Adult Rat Brain

Abstract

Background: Ischemia, white matter injury, and Alzheimer's disease (AD) pathologies often co-exist in aging brain. How one condition predisposes to, interacts with, or perhaps causes the others remains unclear.

Objectives: To better understand the link between ischemia, white matter injury, and AD, adult rats were administered lipopolysaccharide (LPS) to serve as an inflammatory stimulus, and 24 h later subjected to 20-min focal cerebral ischemia (IS) followed by 30-min hypoxia (H).

Methods: Myelin and axonal damage, as well as amyloid-β (Aβ) and amyloid-β protein precursor (AβPP) deposition were examined by Western blot and immunocytochemistry following LPS/IS/H. Findings were compared to the 5XFAD mouse AD brain.

Results: Myelin/axonal injury was observed bilaterally in cortex following LPS/IS/H, along with an increase in IL-1, granzyme B, and LPS. AβPP deposition was present in ischemic striatum in regions of myelin loss. Aβ(1-42) and AβPP were deposited in small foci in ischemic cortex that co-localized with myelin aggregates. In the 5XFAD mouse AD model, cortical amyloid plaques also co-localized with myelin aggregates.

Conclusions: LPS/IS/H produce myelin injury and plaque-like aggregates of myelin. AβPP and Aβ co-localize with these myelin aggregates.

Keywords: Alzheimer’s disease; amyloid plaques; amyloid-β; amyloid-β protein precursor; hypoxia; lipopolysaccharide; myelin; myelin basic protein.

Fig.1

Western blot analysis of brain samples of animals subjected to lipopolysaccharide /ischemia /hypoxia (LPS/IS/H). Animals were subjected to LPS/IS/H and sacrificed 4 weeks, 8 weeks, and 12 weeks later. Naïve animals were used as control. One animal in each time point was examined. The cortex, basal ganglia, and hippocampus ipsilateral to the focal ischemia and contralateral to the ischemia were taken for Western blotting. A) Staining of brain samples was performed using antibodies to myelin basic protein (MBP), interleukin-1β (IL-1β), and Granzyme B (Grzm B). Note decreased MBP in the ipsilateral hemisphere compared to control and contralateral hemisphere; appearance of degraded MBP (dMBP) in the ipsilateral and contralateral hemisphere; and the gradual induction of both IL-1β and Grzm B in the ipsilateral and contralateral hemisphere from 4 to 12 weeks after LPS/IS/H. β-actin was used as the loading control. B) LPS (MW ∼30 kDa) was detected on Western blot analysis in cortex ipsilateral and contralateral to the focal cerebral ischemia at 4 weeks, 8 weeks, and 12 weeks after the LPS/IS/H. Note increased LPS at 12 weeks compared to that at 4 or 8 weeks. LPS was absent in control brain. β-actin was used as loading control, showing somewhat less loading in the 8 week samples.

Fig.2

Western blot analysis for IL-1β and granzyme B in the cortex ipsilateral to ischemia at 12 weeks following LPS/IS/H and saline/IS/H. A) Interleukin-1β (IL-1β) was induced after saline/IS/H and LPS/IS/H. Note that two bands of proteins (around 150 kDa and 120 kDa) were detected following saline/IS/H and only one protein band (around 140 kDa) was detected following LPS/IS/H. B) Granzyme B was not detectable following saline/IS/H but was markedly induced after LPS/IS/H. β-actin was used as the loading control.

Fig.3

MBP staining in control cortex and one week following LPS/IS/H. A) Myelin basic protein (MBP) immunostaining of cortex from a control animal, and double immunostaining for axonal neurofilament light chain protein (NF-L) show that they co-localize for the most part (Merge). B) MBP immunostaining of cortex ipsilateral to the focal ischemia at one week post LPS/IS/H. Note staining in a pattern resembling blood vessels (white arrow) and lighter staining of axons throughout (figure from ipsilateral hemisphere but similar findings bilaterally). C) MBP and caveolin-1 (CVL1) double staining show that MBP is co-localized with endothelial cells (Merge) in cortex at 1 week post LPS/IS/H. Bar = 50 μm.

Fig.4

Tangles of dystrophic myelinated axons in cortex after LPS/IS/H. Myelin basic protein (MBP) immunostaining of cortex ipsilateral to the focal ischemia at 2 weeks (B), 4 weeks (A), 8 weeks (C), and 12 weeks (D) following LPS/IS/H. Note a tangle of enlarged MBP immunostained fibers that are focal and disorganized (A-D, white arrow). MBP staining of axons perpendicular and parallel to the cortical surface is observed which appears to be normal (A, figure from ipsilateral hemisphere but similar findings bilaterally). MBP immunostaining of the fibers in the tangle (E) co-localizes with axonal neurofilament light chain protein (NF-L) (F) as shown by the orange fibers in the merged image (G). These data suggest the tangles are composed of enlarged fibers that appear to be enlarged myelinated axons that are focal and disorganized. Bar = 50 μm.

Fig.5

Localization of N-SMase2 in cortex of control rat and in rats 4 weeks and 12 weeks following LPS/IS/H. In control, N-SMase2 was localized to occasional cell bodies and to occasional axons, with N-SMase2 and neurofilament light chain protein (NF-L) co-localizing in these axons (N-SMase2/NF-L). At 4 weeks post LPS/IS/H, N-SMase2 was localized to large numbers of abnormal linear structures and some of these structures co-localized with NF-L (N-SMase2/NF-L). By 12 weeks post LPS/IS/H, N-SMase2 was again observed in a few scattered axons in cortex that co-localized with NF-L and appeared similar to controls. Bars = 50 μm.

Fig.6

Double staining of myelin basic protein (MBP) with lysosomal-associated membrane protein 1 (Lamp1) in the cortex following LPS/IS/H. At 4 weeks post LPS/IS/H, focal areas of Lamp1 immunostaining and MBP immunostaining were co-localized (Merge). Bar = 50 μm.

Fig.7

Myelin basic protein (MBP)/myelin loss and rodent Aβ deposition (AβPP) in the hemisphere ipsilateral to ischemia. A) Naïve control striatum. MBP immunostaining revealed large axon bundles in control striatum. There was no rodent Aβ (R Aβ) staining in normal brain including striatum. B) Striatum at 8 weeks post LPS/IS/H showed loss of normal MBP staining, and with deposition of R Aβ (which is likely AβPP protein) in these areas of MBP loss. Bars = 50 μm. C) Western blot analysis for R Aβ at 12 weeks following saline/IS/H and LPS/IS/H. A 100 kDa protein band was detected on Western blot for R Aβ either following saline/IS/H or following LPS/IS/H. Given this 100 kDa molecular weight, this protein is probably AβPP. β-actin was used as the loading control. D) The expression of R Aβ/AβPP following LPS/IS/H was significantly increased compared to that following saline/IS/H.  ^**p <  0.01.

Fig.8

Western blot analysis for amyloid-β protein precursor (AβPP) in the hemisphere ipsilateral to ischemia at 12 weeks following LPS/IS/H and saline/IS/H compared to naïve control. A) A 100 kDa molecular weight protein was detected on Western blot analysis for AβPP either following saline/IS/H or following LPS/IS/H and in the naïve control. B) The expression of AβPP decreased in saline/IS/H animals compared to naïve, and LPS/IS/H increased AβPP compared to saline/IS/H. The expression of AβPP following LPS/IS/H was not significantly different compared to naïve controls. β-actin was used as the loading control.  ^***p <  0.001.

Fig.9

Co-localization of myelin aggregates with rodent Aβ and Aβ_1 - 42 expression in the hemisphere ipsilateral to ischemia at 12 weeks following LPS/IS/H. A) Myelin aggregates stained with myelin basic protein (MBP) co-localized with rodent Aβ (R Aβ) deposits. Most of the MBP stained foci co-localized with rodent Aβ (white arrows, Merge) whereas some did not (yellow arrows). R Aβ likely represents AβPP. B) Myelin associated glycoprotein (MAG) co-localized with Aβ_1 - 42 deposits. MAG immunostained foci in cortex co-localized with Aβ_1 - 42 (B, Merge). Bars = 50 μm. C) Western blot analysis for Aβ_1 - 42 at 12 weeks following saline/IS/H and LPS/IS/H. Two bands of ∼150 kDa and ∼140 kDa were detected on Western blots using the Aβ_1 - 42 antibody which showed marked induction of both bands following LPS/IS/H compared to saline/IS/H. D) Quantification of the expression of Aβ_1 - 42 showed that it markedly increased following LPS/IS/H compared to that following saline/IS/H.  ^**p <  0.01.

Fig.10

Western blot analysis for Aβ_1 - 40/42 in the hemisphere ipsilateral to ischemia at 12 weeks following LPS/IS/H and saline/IS/H compared to naïve control. A) A protein with molecular weight as 150 kDa was detected on Western blot analysis for Aβ_1 - 40/42 either following saline/IS/H or following LPS/IS/H and in naïve control. B) The expression of Aβ_1 - 40/42 decreased following saline/IS/H compared to naïve, and Aβ_1 - 40/42 increased following LPS/IS/H compared to saline/IS/H. The expression of Aβ_1 - 40/42 following LPS/IS/H was not significantly different from naïve controls. β-actin was used as the loading control.  ^***p <  0.001.

Fig.11

Double staining of myelin basic protein (MBP) and (E, E)-1-fluoro-2,5-bis (3-hydroxycarbonyl-4-hydroxy) styrylbenzene (FSB) in the amyloid plaques of 5XFAD mice. MBP positive myelin aggregates were detected in cortex of 8 month (8M, left panel) old (A) and 10 month (10M, left panel) old (b) 5XFAD mice. These MBP myelin aggregates co-localized (Merge) with FSB stained amyloid plaques in the cortex of 8 month old (8M, middle upper panel) and 10 month (10M, middle lower panel) old 5XFAD mice. MBP aggregates at 10 months (10M) seemed somewhat fragmented (B). The FSB stained plaques co-localized with MBP staining in 8M (upper right panel) and 10M (lower right panel) old animals (Merge). Bar = 50 μm.