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. 2007 Nov 21;2(11):e1205.
doi: 10.1371/journal.pone.0001205.

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS

Svitlana Garbuzova-Davis  1 , Samuel Saporta, Edward Haller, Irina Kolomey, Steven P Bennett, Huntington Potter, Paul R Sanberg
Affiliations

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS

Svitlana Garbuzova-Davis et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characteristics of disease progression in G93A mice.
(A) Body weight and (B) extension reflex of G93A and control C57BL/6J mice. G93A mice at about 13 weeks of age showed initial signs of disease such as weight loss and reduced hindlimb extension. Terminal stage of disease was observed at 17–18 weeks of age, as demonstrated by complete hindlimb paralysis, significant reduction of body weight and absence of hindlimb extension. Arrows indicate the age of mice when euthanatasia was performed. The five pointed star in A indicates difference (p = 0.06) in body weights between G93A mice at 13 weeks of age and 18 weeks of age; the four pointed star indicates a significant difference (p = 0.007) in body weights between G93A and C57BL/6J mice at 18 weeks of age. The four pointed star in B indicates a significant difference in extension reflex (p<0.001) between G93A mice at 13 weeks of age and 18 weeks of age.
Figure 2
Figure 2. Motor neurons in the cervical spinal cord of G93A mice at early and late stage of disease (cresyl violet staining).
In the cervical spinal cord, many healthy motor neurons with large soma and neuritic processes were identified in the control C57BL/6J mice at (A) 12–13 weeks of age and (B) 19–20 weeks of age. In G93A mice, numerous motor neurons with vacuolization (asterisks) were found at (C) 13 weeks of age and (D) decreased numbers of motor neurons were noted in 17–18 week old mice. Motor neurons of various sizes displayed vacuolization (asterisks). Scale bar on left side is 200 µm, right side is 50 µm.
Figure 3
Figure 3. Motor neurons in the lumbar spinal cord of G93A mice at early and late stage of disease (cresyl violet staining).
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.
Figure 4
Figure 4. Evans Blue fluorescence in the cervical spinal cord of G93A mice at early and late stages of disease.
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.
Figure 5
Figure 5. Evans Blue fluorescence in the lumbar spinal cord of G93A mice at early and late stages of disease.
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.
Figure 6
Figure 6. Immunofluorescence staining for laminin in the cervical spinal cord of G93A mice at early and late stages of disease.
Many blood vessels of different diameter were immunoreactive for laminin-1 (red) in the control C57BL/6J mice at (A) 12–13 weeks of age and (B) 19–20 weeks of age. In G93A mice at (C) initial or (D) late stages of disease, capillaries appear to be less numerous. In some early symptomatic G93A mice, (C) blurry spots around capillaries were found. 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.
Figure 7
Figure 7. Immunofluorescence staining for laminin in the lumbar spinal cord of G93A mice at early and late stages of disease.
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.
Figure 8
Figure 8. Immunofluorescence staining for Glut-1 in the cervical and lumbar spinal cords of G93A mice at early and late stages of disease.
Cervical spinal cord. 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. Lumbar spinal cord. 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.
Figure 9
Figure 9. Immunohistochemical staining for endothelial cells (CD146) and astrocytes (GFAP) in the cervical spinal cord of G93A mice at early and late stages of disease.
(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.
Figure 10
Figure 10. Immunohistochemical staining for endothelial cells (CD146) and astrocytes (GFAP) in the lumbar spinal cord of G93A mice at early and late stages of disease.
(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.
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