Evans Blue fluorescence in the cervical spinal cord of G93A mice at early and late stages of disease.

<p>In the cervical spinal cord, EB was clearly detected within the blood vessels (red, arrowheads) in the control C57BL/6J mice at (A, B, C) 12–13 weeks of age or (D, E) in the lumen of vessels (brilliant green) at 19–20 weeks of age. In G93A mice, vascular leakage of EB (red, arrows) was detected (F, G) at early (13 weeks of age) disease symptoms and (H, I, J) at end-stage of disease (17–18 weeks of age) when more EB extravasation was seen. Arrowheads in F and I indicate vessel permeability. Scale bar in A–J is 25 µm.</p

Evans Blue fluorescence in the lumbar spinal cord of G93A mice at early and late stages of disease.

<p>In the lumbar spinal cord, EB dye (red, arrowheads) was determined intravascularly in the control C57BL/6J at (A, B) 12–13 weeks of age and (C, D) 19–20 weeks of age similar to the cervical spinal cord. EB extravasation abnormalities were found in G93A mice at (E, F) 13 weeks of age (red, arrows). (G, H) Significant EB diffusion (red, arrows) into the parenchyma of the lumbar spinal cord from many blood vessels was detected in G93A mice at end-stage of disease (17–18 weeks of age). Arrowheads in F and G indicate vessel permeability. Scale bar in A–H is 25 µm.</p

Immunofluorescence staining for Glut-1 in the cervical and lumbar spinal cords of G93A mice at early and late stages of disease.

<p><i>Cervical spinal cord.</i> High expression of Glut-1 (red) was determined in endothelial lining of many blood vessels of various diameters in the cervical spinal cord of the control C57BL/6J mice at (A), (B) 12–13 weeks of age and (C) 19–20 weeks of age. In G93A mice at (D), (E) initial or (F), (G) late stages of disease, immunoreaction for Glut-1 in the endothelial cells appear to be low, or nonexistent. The nuclei in A–G are shown with DAPI. Scale bar in A and E is 50 µm; B, C, D, F, G is 25 µm. Outline of white dots indicates configuration of blood vessels. <i>Lumbar spinal cord.</i> Similar to the cervical spinal cord, most Glut-1-positive endothelial cells (red) were observed in the control C57BL/6J mice at (H), (I) 12–13 weeks of age and (J) 19–20 weeks of age. Less Glut-1 expression was found in G93A mice at (K), (L) early or (M), (N) end-stage of disease. The nuclei in H–N are shown with DAPI. Scale bar in J, M, N is 50 µm; H, I, K, L is 25 µm.</p

Immunofluorescence staining for laminin in the lumbar spinal cord of G93A mice at early and late stages of disease.

<p>Various laminin-positive vessels (red) were observed in the control C57BL/6J mice at (A) 12–13 weeks of age and (B) 19–20 weeks of age similar to cervical spinal cord results. Fewer blood vessels were labeled in G93A mice at (C) early or (D) end-stage of disease. The nuclei in A–D are shown with DAPI. Scale bar in A, B, C, D is 200 µm; inserts a, b, c, d is 50 µm.</p

Immunohistochemical staining for endothelial cells (CD146) and astrocytes (GFAP) in the lumbar spinal cord of G93A mice at early and late stages of disease.

<p>(A, B, C) Similar to cervical spinal cord, endothelial cells (green, arrowheads) and astrocytes (red, asterisk) in C57BL/6J mice at 19–20 weeks of age appeared normal. In G93A mice at (D, E) early or (F, G) end-stage of disease, decreased CD146 antigen expression by endothelial cells (green, arrowheads) was observed. Note: increased astrocyte activation in the lumbar spinal cord (F, G, asterisks) was detected in G93A mice at late stage of disease. The nuclei in A, C, D, and F are shown with DAPI. Scale bar in A, C, D, F is 50 µm; B, E, G is 25 µm.</p

Immunohistochemical staining for endothelial cells (CD146) and astrocytes (GFAP) in the cervical spinal cord of G93A mice at early and late stages of disease.

<p>(A, B) Normal appearance of endothelial cells (green, arrowheads) and delineated astrocytes (red, asterisk) was observed in the control C57BL/6J mice at 19–20 weeks of age. Endothelia (green, arrowheads) surrounding capillaries were partially revealed in G93A mice at (C, D) initial or (E, F) late stages of disease. Note: increased astrocyte activation in the cervical spinal cord (F, asterisks) was detected in G93A mice at late stage of disease. The nuclei in A, C, and E are shown with DAPI. Scale bar in A, C, E is 50 µm; B, D, F is 25 µm.</p

Motor neurons in the lumbar spinal cord of G93A mice at early and late stage of disease (cresyl violet staining).

<p>In the lumbar spinal cord, C57BL/6J mice at (A) 12–13 weeks of age and (B) 19–20 weeks of age showed numerous motor neurons with strong Nissl body staining. Most degenerated or swollen motor neurons (asterisks) were found in G93A mice at (C) early (13 weeks of age) and (D) late (17–18 weeks of age) stages of disease; most surviving motor neurons were small. Scale bar on left side is 200 µm, right side is 50 µm.</p