Figure 1.
Cytokine and chemokine levels in the CNS of Twitcher mice.
The fold-elevation of various cytokines/chemokines in the brain is shown. Among all the assayed molecules, the chemokine KC showed the greatest fold change in the brains (>15-fold increase) of the Twitcher mice (A). The levels of KC in the brains (B) and the spinal cords (C) of the Twitcher mice showed a progressive increase with time. The vertical bars represent the means and the error bars represent one SEM; **p<0.01, ***p<0.001.
Figure 2.
Flow cytometric characterization of inflammatory cells in the KC−/−GALC−/− brains.
The upper row of Panel A contains representative bivariate contour plots showing CD8 and CD4 T-cells at day 36 in various groups of mice. The lower row in panel A contain representative bivariate contour plots showing activated microglia (CD45hi CD11b+, upper right quadrant) isolated from the brain at 36 days of age. There is no significant increase in CD8+ T-cells in KC−/−GALC+/+, KC+/+GALC−/− or KC−/−GALC−/− mice compared to the KC+/+GALC+/+ mice (B). There is no significant difference in the CD4+ T-cells in the brains of the KC−/−GALC−/− and KC+/+GALC−/− mice compared to the KC+/+GALC+/+ mice (C). There is no significant difference between the KC+/+GALC−/− mice and KC−/−GALC−/− mice in the number of activated microglia (D).
Figure 3.
LFB/PAS staining of Twitcher mice lacking KC or CXCR2.
Histology of the brains showing LFB staining (blue) and PAS staining (pink) in the corpus callosum (A-D) and cerebellum (E-H) in the wildtype, Twitcher, KC−/−GALC−/− mice and CXCR2−/−GALC−/− mice. The KC−/−GALC−/− forebrain (C) and cerebellum (G) show histology which is essentially identical to the Twitcher mice (KC+/+GALC−/−; B and F) with similar myelin staining and distribution of globoid cells. The CXCR2−/−GALC−/− mouse brains also demonstrate similar histology (D, H) compared to the Twitcher mice (B, F). There is no apparent difference between the KC+/+GALC+/+ and KC−/−GALC+/+ or the CXCR2−/−GALC+/+ mice in all the sections examined (images not shown). Scale bar equals 50 µm.
Figure 4.
Oligodendrocyte precursor proliferation in the lumbar spinal cord of twitcher mice lacking KC or CXCR2.
Arrowheads in panels A-D identify oligodendrocyte precursors expressing NG2 (red) with a more dendritic morphology. Arrowheads also identify the regions highlighted in the higher magnification insets. Minimal BrdU staining (green) was noted in wildtype mice (A) (scale bar, 25 µm). In B, C and D, the arrowheads identify proliferating oligodendrocytes that co-stain for NG2 (red) and BrdU (green). Blue represents nuclear staining with DAPI. There is no statistically significant difference in the total number of NG2+ cells in the spinal cords of various groups (E). A statistically significant (*** p<0.001) increase in number of NG2+BrdU+ cells/field is seen in the Twitcher and CXCR2−/− GALC−/− spinal cords compared to WT mice (F). There is no significant difference in the number of NG2+BrdU+ cells in KC−/−GALC−/− mice compared to Wildtype, Twitcher, or CXCR2−/− GALC−/− groups.
Figure 5.
Effect of CXCR2 and KC bone marrow chimeras on the progression of GLD.
The survival of Twitcher mice transplanted with CXCR2-deficient bone marrow (green, GALC+/+CXCR2−/− to Twitcher) or KC−/−GALC−/− mice transplanted with wild type bone marrow (black, wildtype to KC−/−GALC−/−) is not significantly different from Twitcher mice receiving wild type bone marrow (blue, wildtype to twitcher) (A). Twitcher mice transplanted with Twitcher bone marrow (red, Twitcher to Twitcher) have a similar survival (median age = 45 days) compared to untransplanted Twitcher mice (data not shown). The weights of various bone marrow chimeras are not significantly different from each other, but are significantly decreased compared to the wild type controls (B).
Figure 6.
Altered cytokine and growth factor levels in the spinal cord of Twitcher mice that could possibly compensate for the lack of KC or CXCR2.
MIP-2 (CXCL2) (A), PDGF-BB (B), and FGF-2 (C) levels are significantly and progressively elevated in the spinal cords of the Twitcher mice. Vertical bars represent the mean and the error bars represent one SEM (**p<0.01, ***p<0.001).