Evidence of Compromised Blood-Spinal Cord Barrier in Early and Late Symptomatic SOD1 Mice Modeling ALS

Background: The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease. Methodology/Principal Findings: Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage. Conclusions/Significance: Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease

Ultrastructure of Blood-Brain Barrier and Blood-Spinal Cord Barrier in SOD1 Mice Modeling ALS

The purpose of this study was to determine the ultrastructure of the blood–brain barrier (BBB) and blood–spinal cord barrier (BSCB) in G93A SOD1 mice modeling ALS at different stages of disease. Electron microscope examination of brainstem, cervical and lumbar spinal cords was performed in ALS mice at early and late stages of disease. Our results show disorganized mitochondrial cristae and degenerating mitochondria in endothelial cells and neuropil, swollen astrocyte foot processes, swollen and degenerating capillary endothelial cells, astrocytes and motor neurons and extensive extracellular edema. In spite of progressive extracellular edema in neural tissue, capillary endothelial cell tight junctions appeared to remain intact in early and late symptomatic animals. Results show that disruption of BBB and BSCB was evident in areas of motor neuron degeneration in G93A mice at both early and late stages of disease. Capillary rupture was observed in brainstem in early symptomatic G93A mice. Capillary ultrastructure revealed that endothelial cell membrane and/or basement membrane damage occurred, followed by vascular leakag

Evidence of Compromised Blood-Spinal Cord Barrier in Early and Late Symptomatic SOD1 Mice Modeling ALS

Background: The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease. Methodology/Principal Findings: Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage. Conclusions/Significance: Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease

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 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

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

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