All animal protocols were approved by the University of Michigan Committee on the Use and Care of Animals. A total of 25 male wild-type (WT) C57/BL6 mice (22–30g; Charles River Laboratories) and 11 male lipocalin 2 knockout (LCN2−/−) mice (University of Michigan Breeding Core, gift from Dr. Xiaoli Chen, University of Minnesota) were used. SAH was induced using an endovascular perforation technique as previously described (WT, n=19; LCN2−/−, n=6).3 (link), 5 (link) Sham control mice underwent the same surgical procedure without perforation (WT, n=6; LCN2−/−, n=5). MRI was performed 24 hours after SAH in a 7.0-T Varian MR scanner with acquisition of T2 fast spin-echo and T2* gradient-echo sequences using a field of view of 20×20 mm, matrix of 256×256 mm, and 25 coronal slices (0.5 mm thick). To calculate the volume, the area of white matter T2 hyperintensity was measured in all slices and multiplied by section thickness. Ventricular volume was measured as previously described.6 (link)After MRI, mice were euthanized and SAH severity assessed using a modified grading system.3 (link) For immunohistochemistry, the forebrain was embedded and sliced into 10-μm-thick coronal sections (Sham, n=4; WT+SAH, n=6; LCN2−/−+SAH, n=5). LCN2 (dilution 1:200; R&D systems), glial fibrillary acidic protein (GFAP, 1:400; Millipore), Iba-1 (1:200; Wako), NG2 (1:200; Millipore), β-amyloid precursor protein (β-APP, 1:1000; Invitrogen), and degraded myelin basic protein (DMBP, 1:2000; Millipore) antibodies were used. NG2 is an oligodendrocyte precursor cell marker. NG2 expression correlated with axonal degeneration and oligodendrocyte death.7 (link) β-APP and DMBP identify damaged axons and degraded myelin, respectively. For quantification, 4 slides from each brain with each slide containing 3 fields from white matter tract (corpus callosum, external capsule, or fimbriae) were digitized using a BX-51 Olympus microscope (40× objective). The number of NG2 positive cells was counted. β-APP and DMBP immunoreactivity were scored 0–3 (none-extensive) as described previously,8 (link) and the score summated over 12 fields. Image analysis was performed by blinded investigator using Image J software.
Data are expressed as means±SD. Statistical differences among groups were analyzed using one-way ANOVA, Spearman rank correlation test, and Mann-Whitney U test. A Bonferroni correction was used for multiple comparisons. P<0.05 was considered statistically significant.
Data are expressed as means±SD. Statistical differences among groups were analyzed using one-way ANOVA, Spearman rank correlation test, and Mann-Whitney U test. A Bonferroni correction was used for multiple comparisons. P<0.05 was considered statistically significant.
