Electron microscope examination of the cerebellum of <i>Naglu</i> mutant mice.

<p>(<b>A</b>), (<b>B</b>) Similar to ultrastructure of microvessels in the cerebral cortex, hippocampus, and striatum, the intact BBB in the cerebellum of a C57 BL/6J control mouse consists of an endothelial cell and a single layer of basement membrane. (<b>C</b>) Edematous space around vessel as well as vacuolated pericyte is found in the cerebellum of 3 months old mutant mouse. Vacuoles are also seen in the cytoplasm of neuron located near vessel. (<b>D</b>) In 6 months old <i>Naglu</i> mice, two highly vacuolated perivascular macrophages surround a microvessel. Edematous space around vessels is in contact with astrocytic end-feet. (<b>E</b>) Pericytes, which contain large vacuoles displacing and destroying cell organelles, can be seen under the capillary basement membrane in mouse at the same stage of disease. Large vacuoles are also visible outside vessel. <b>EC</b> - endothelial cell, <b>BM</b> - basement membrane, <b>E</b> – erythrocyte, <b>m</b> – mitochondrion, <b>A</b> – axon, <b>N</b> – neuron, <b>P</b> – pericyte, <b>PM</b> – perivascular macrophage, <b>Nu</b> – nucleus, <b>V</b> – vacuole, <b>></b> - extracellular edematous space. Magnifications: (<b>A</b>) 7,100x; (<b>C</b>), (<b>D</b>): 4,400x; (<b>B</b>): 28,000x; (<b>E</b>): 11,000x.</p

Electron microscope examination of the cerebral cortex of <i>Naglu</i> mutant mice.

<p>(<b>A</b>) Representative area of C57 BL/6J control mouse cerebral cortex characterized by normal ultrastructural appearance of neurons, myelinated axons, and capillaries. (<b>B</b>) Capillary at high magnification from the cerebral cortex of control mouse consists of a single layer of endothelial cells surrounded by a layer of basement membrane, forming an intact BBB. Erythrocytes can be observed in the lumen of the capillary. Organelles in all cells were well preserved. (<b>C</b>) In the cortex of <i>Naglu</i> mutant mouse at 3 months of age (early symptomatic), endothelial cells and pericyte show formation of numerous large vacuoles in their cytoplasm. Microvilli and their fragments can be observed floating free in the capillary lumen. (<b>D</b>) Highly vacuolated pericyte was found in contact with microvessel basement membrane in 3 months old mutant mice. Edematous space is observed around vessels. (<b>E</b>) In late symptomatic (6 months of age) mutant mouse, large vacuoles containing light and dark material cause swelling of a pericyte, displacing organelles. (<b>F</b>) Longitudinal section of capillary showing large perivascular macrophage with numerous vacuoles in its cytoplasm near the blood vessel in the cortex of a 6 month old <i>Naglu</i> mouse. Numerous vacuoles were found in cells surrounding the capillary. <b>EC</b> - endothelial cell, <b>BM</b> - basement membrane, <b>E</b> – erythrocyte, <b>m</b> – mitochondrion, <b>A</b> – axon, <b>P</b> – pericyte, <b>PM</b> – perivascular macrophage, <b>Nu</b> – nucleus, <b>V</b> – vacuole, <b>L</b> – lysosome, <b>asterisks</b> - microvilli. Magnifications: (<b>A</b>), (<b>D</b>), (<b>F</b>): 7,100x; (<b>B</b>), (<b>C</b>): 14,000x; (<b>E</b>): 4,400x.</p

Pattern of vascular leakage in the brains of <i>Naglu</i> mice at different stages of disease.

<p><b>Key: Cx</b> – cerebral cortex, <b>Hip</b> – hippocampus, <b>Cb</b> – cerebellum, <b>Md</b> – medulla, <b>MB</b> – midbrain, <b>OB</b> – olfactory bulb, <b>PS</b> – pons, <b>ST</b> – striatum, <b>TH</b> – thalamus, <b>EB</b> – Evans Blue, <b>Alb</b> – albumin, <b>--</b> - no leak, + - moderate leak (near abluminal side of the capillaries), <b>++</b> - significant leak (at a distance from the capillaries). Note: no vascular leakage was determined in examined brain blood vessels from control mice at 3, 6 or 10–12 months of age.</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

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

Electron microscope examination of the striatum of <i>Naglu</i> mutant mice.

<p>(<b>A</b>) Structural integrity of capillaries in the striatum was normal in C57 BL/6J control mice. (<b>B</b>) A single layer of endothelial cells surrounded by a layer of basement membrane in control mouse seen at high magnification. (<b>C</b>), (<b>D</b>) Edematous space and large vacuoles are indicated around vessels. Vacuolated pericytes can be seen in the striatum of early symptomatic <i>Naglu</i> mice. (<b>E</b>) Large vacuoles were found in endothelial cells and pericytes of late symptomatic animals. (<b>F</b>) A high magnification image of a capillary from the striatum of a 6 month old mutant mouse showing an endothelial cell with endoplasmic reticulum swelling and formation of a large vacuole in its cytoplasm. Edematous space has appeared around the capillary. Multiple layers of endothelial cells and basement membrane can be observed here, indicating a reparative process taking place. The luminal endothelial cells are damaged<b>. EC</b> - endothelial cell, <b>BM</b> - basement membrane, <b>E</b> – erythrocyte, <b>m</b> – mitochondrion, <b>A</b> – axon, <b>P</b> – pericyte, <b>PM</b> – perivascular macrophage, <b>Nu</b> – nucleus, <b>V</b> – vacuole, <b>+</b> - swollen endoplasmic reticulum, <b>asterisks</b> - microvilli, <b>></b> - extracellular edematous space. Magnifications: (<b>A</b>), (<b>C</b>): 7,100x; (<b>B</b>), (<b>E</b>): 14,000x; (<b>D</b>): 8,900x; (<b>F</b>): 28,000x.</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

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

Evans Blue (EB) fluorescence in the brains of <i>Naglu</i> mice.

<p>In the brain, EB can be clearly detected within the blood vessels (<b>A</b>, <b>B</b>, <b>C</b>, red, arrowheads) in the control C57 BL/6J mouse at 12 months of age. In <i>Naglu</i> mice, vascular leakage of EB (red, arrows) is visible in various brain structures (<b>D</b>, <b>E</b>, <b>F</b>) at early (3 months of age), (<b>J</b>, <b>H</b>, <b>I</b>) at late (6 months of age), and (<b>J</b>, <b>K</b>, <b>L</b>) at end stage of disease (10 months of age). Significant EB diffusion into the brain parenchyma from many blood vessels can be detected in end-stage <i>Naglu</i> mouse (10–12 months of age). <b>Cx</b> – cerebral cortex, <b>Hip</b> – hippocampus, <b>Crb</b> – cerebellum. Scale bar in A through L 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