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 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

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

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

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

<p>(<b>A</b>), (<b>B</b>) Hippocampal area of C57 BL/6J control mouse shows typical normal ultrastructure of neurons and all capillary components. (<b>C</b>) Vacuolated endothelial cells and pericytes can be observed in 3 months old <i>Naglu</i> mouse. A highly vacuolated perivascular macrophage was also noted. Platelets attracted to a region of endothelial cell damage are visible. (<b>D</b>) Perivascular space can be observed around the capillaries in the hippocampus of an early symptomatic mouse at 3 months of age. (<b>E</b>) In <i>Naglu</i> mouse at 6 months of age, a microaneurysm is visible in the hippocampus, adjacent to a ruptured endothelium. In the microaneurysm, an erythrocyte can be seen trapped under the basement membrane, portending imminent aneurysm rupture. (<b>F</b>) Under high magnification, distended nucleus of pericyte with large vacuoles can be observed in late symptomatic mutant mouse. <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>Ma</b> – microaneurysm, <b>pl</b> – platelets, <b>asterisks</b> - microvilli, <b>></b> - extracellular edematous space. Magnifications: (<b>A</b>), (<b>B</b>), (<b>D</b>), (<b>E</b>): 7,100x; (<b>C</b>): 3,500x; (<b>F</b>): 8,900x.</p

Immunohistochemical staining for GM3 ganglioside in the brains of <i>Naglu</i> mice.

<p>In the brain of control C57 BL/6J mouse at 12 months of age, GM3 ganglioside is not detected within the vascular endothelium of blood vessels (<b>A</b>, <b>B</b>, <b>C</b>, arrowheads). In early symptomatic (3 month of age) <i>Naglu</i> mice, some endothelial cells stained positive for GM3 (<b>D</b>, <b>E</b>, <b>F</b>, arrows) and some cells were negative for GM3 (<b>D</b>, <b>E</b>, <b>F</b>, arrowheads). GM3 accumulation (arrows) was seen in the endothelia of numerous brain blood vessels (<b>G</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 stages of disease (10 months of age). Significant GM3 ganglioside accumulation was also seen in neurons (asterisks) in <i>Naglu</i> mice. <b>Cx</b> – cerebral cortex, <b>Hip</b> – hippocampus, <b>Crb</b> – cerebellum. Scale bar in A through I is 25 µm.</p