White matter damage and the effect of matrix metalloproteinases in type 2 diabetic mice after stroke

Abstract

Background and purpose: Diabetes mellitus leads to a higher risk of ischemic stroke and worse outcome compared to the general population. However, there have been few studies on white matter (WM) damage after stroke in diabetes mellitus. We therefore investigated WM damage after stroke in mice with diabetes mellitus.

Methods: BKS.Cg-m(+/+)Lepr(db)/J (db/db) type 2 diabetes mellitus mice and db(+) non-diabetes mellitus mice were subjected to middle cerebral artery occlusion. Functional outcome, immunostaining, zymography, Western blot, and polymerase chain reaction were used.

Results: After stroke, mice with diabetes mellitus exhibited significantly increased lesion volume and brain hemorrhagic and neurological deficits compared to mice without diabetes mellitus. Bielshowsky silver, luxol fast blue, amyloid precursor protein, and NG2 expression were significantly decreased, indicating WM damage, and matrix metalloproteinase (MMP)-9 activity was significantly increased in the ischemic brain of mice with diabetes mellitus. Subanalysis of similar lesions in mice with and without diabetes mellitus demonstrated mice with diabetes mellitus had significantly increased WM damage than in mice without diabetes mellitus (P<0.05). To investigate the mechanism underlying diabetes mellitus-induced WM damage, oxygen-glucose deprivation-stressed premature oligodendrocyte and primary cortical neuron cultures were used. High glucose increased MMP-2, MMP-9, cleaved caspase-3 levels, and apoptosis, as well as decreased cell survival and dendrite outgrowth in cultured primary cortical neuron. High glucose increased MMP-9, cleaved caspase-3 level, and apoptosis, and decreased cell proliferation and cell survival in cultured oligodendrocytes. Inhibition of MMP by GM6001 treatment significantly decreased high glucose-induced cell death and apoptosis in cultured primary cortical neuron and oligodendrocytes but did not alter dendrite outgrowth in primary cortical neuron.

Conclusions: Mice with diabetes mellitus have increased brain hemorrhage and show more severely injured WM than mice without diabetes mellitus after stroke. MMP-9 upregulated in mice with diabetes mellitus may exacerbate WM damage after stroke in mice with diabetes mellitus.

Figure 1

Neurological outcome and Lesion volume after stroke. A, Hematoxylin and eosin staining in mice with and without diabetes mellitus (DM). B, Lesion volume and brain hemorrhage measurements. C, Modified neurological severity score and foot-fault tests after stroke in mice with and without DM (n=11 per group).

Figure 2

Diabetes mellitus (DM) increased white matter (WM) and axon damage in the ischemic brain compared to mice without DM. Immunostaining of luxol fast blue (dark blue; A), NG2 (brown; B), Bielschowsky silver (black; C), and amyloid precursor protein (brown; D) and quantification data in the ischemic boundary zone in control mice with and without DM (n=11 per group). Scale bar in A–D=100 μm.

Figure 3

Subanalysis in similar lesion animals. Quantification data in the ischemic boundary zone for luxol fast blue (A), NG2 (B), and Bielschowsky sliver (C) staining in similar lesion of mice with and without diabetes mellitus (n=5 per group).

Figure 4

Diabetes mellitus (DM) increased matrix metalloproteinas expression and activity in the ischemic brain compared to mice without DM after stroke. Real-time polymerase chain reaction (A), Western blot (B), and zymography (C) assays in the ischemic brain in groups with and without DM (n=4 per group).

Figure 5

High glucose (HG) increased cell death and matrix metalloproteinase (MMP) expression in cultured primary cultured neurons (PCN) and oligodendrocytes. Inhibition of MMP attenuated HG-induced cell death in cultured PCN and oligodendrocytes. A and C, Lactate dehydrogenase assay and real-time polymerase chain reaction in cultured PCN (A) and oligodendrocyte (C; n=6 pe group). B, Western blot and quantitative data in PCN and oligodendrocyte (n=4 per group). C, MTS assay in cultured oligodendrocyte (n=6 per group).

Figure 6

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining in cultured primary cultured neurons (PCN) and oligodendrocyte. High gluscose (HG) decreased neurite outgrowth in cultured PCN. Inhibition of matrix metalloproteinase did not attenuate HG-induced neurite outgrowth damage. TUNEL staining and quantitative data in PCN (A) and oligodendrocyte (B). C, TUJ1 immunostaining and neurite outgrowth quantitative data in cultured PCN (red, TUJ1; blue, DAPI; n=6 per group).