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

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 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

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

Cytokine profile in the spleen of G93A mice administered with different MNC hUCB cell doses.

<p>The RNase protection assay was used to determine the mRNA expression of proinflammatory cytokines (IL-1 α, IL-1 β, TNF α, TNF β, and IL-2) and anti-inflammatory cytokine IL-10 in the spleen of G93A mice administered with different MNC hUCB cell doses. Control groups were Media, CsA, hTgn, and C57BL/6 mice. The mRNA expression presented as the optical density (OD) values obtained from each band normalizing against the OD obtained from the L32, a house-keeping gene, band. Lines indicated significant differences (p<0.001, p<0.01, and p<0.05) between mouse groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002494#s2" target="_blank">Results</a>).</p

Cytokine profile in the lumbar spinal cord, brainstem, and motor cortex of G93A mice administered with different MNC hUCB cell doses.

<p>The RNase protection assay was used to determine the mRNA expression of proinflammatory cytokines IL-1 α, IL-1 β, TNF α, and TNF β in the lumbar spinal cord (left column), brainstem (center column), and motor cortex (right column) of G93A mice administered with different MNC hUCB cell doses. Control groups were Media, CsA, hTgn, and C57BL/6 mice. The mRNA expression presented as the optical density (OD) values obtained from each band normalizing against the OD obtained from the L32, a house-keeping gene, band. Lines indicated significant differences (p<0.001, p<0.01, and p<0.05) between mouse groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002494#s2" target="_blank">Results</a>).</p

Human Th1/Th2 cytokines in plasma from G93A mice administered with different MNC hUCB cell doses.

<p>FAST Quant microspot assay for human Th1/Th2 Cytokines (IL-1 β, TNF α, INF γ, IL-2, IL-4, IL-5, IL-6, IL10, and IL-13) in plasma from G93A mice receiving three different cell doses was performed by Schleicher & Schuell BioScience, Inc. (Keene, NH, USA). Plasma samples from hUCB (n = 10) were used as controls. Quantitative results of cytokines were presented as pg/mL. The 50% of mice receiving 25×10⁶ cells showed evidence of human IL-4 and IL-13 (Th2). In 62.5% of mice with 50×10⁶ cell treatment, all human cytokines were detected except for IL-5, IL-5, and IL-10 (Th2). There were no human cytokines detected in mice treated with 10×10⁶ cell dose. The hUCB plasmas themselves, mostly contained Th2 cytokines (IL-4, IL-10, and IL-13).</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

Immunochistochemical staining of MNC hUCB cells in the lumbar spinal cord of G93A mice administered with different cell doses.

<p>MNC hUCB cells were found in the lumbar spinal cord of mice receiving A), B) 10×10⁶; C), D) 25×10⁶; and E), F) 50×10⁶ cells by anti-human nuclei staining (green, asterisks). Merged images are with DAPI. Some a), b), c), d), e), f) MNC hUCB cells expressed Nestin (red, asterisks). Cells in images a, b, c, d, e, f are same in images A, B, C, D, E, F. Scale bar: A–e is 25 µm.</p