Conceived and designed the experiments: SG. Performed the experiments: SG. Analyzed the data: SG. Contributed reagents/materials/analysis tools: SG. Wrote the paper: SG. Other: Consulted with about pathological mechanisms in ALS: PS. Consulted in discussion of findings: HP. Assisted in determining disease stage in ALS mice: SB. Assisted in performing immunohistochemistry: IK. Assisted in performing experiments with Evans Blue extravation: EH. Consulted on experimental design: SS.
The authors have declared that no competing interests exist.
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.
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.
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.
The central nervous system (CNS) is an immunologically privileged zone, which is normally protected from entry of immune cells and serum proteins by the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB). These barriers are specialized structures of the CNS that control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment (reviewed in
Impairment of the BBB occurs in various pathological CNS conditions. Accumulation of collagen in vascular basement membranes and focal necrotic changes in endothelial cells were found in Alzheimer's patients
Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease affecting motor neurons in the spinal cord, motor cortex and brainstem that leads to paralysis and death within five years of disease onset
Additionally, significantly increased levels of albumin, IgG, and C3c have been noted in the cerebrospinal fluid of ALS patients
The G93A mice were monitored weekly for initial disease symptoms and disease symptom progression by body weight and extension reflex, beginning at 7 weeks of age. Body weights of G93A mice gradually increased until 11 weeks of age and then stabilized for 2 weeks (12–13 weeks of age). Tremor, an initial sign of disease, was seen in some mice as early as 13 weeks of age. After this time, mice consistently lost body weight due to muscle atrophy. By 18 weeks of age, G93A mice had lost approximately 14% (p = 0.06) of their 13-weeks-of-age body mass, dropping from 25.4±0.38 g to 21.8±0.55 g (
(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.
Nissl body staining was performed to identify motor neuron condition in the spinal cords of G93A mice in early and late stages of disease. In control C57BL/6J mice, many healthy motor neurons with large soma and neuritic processes were visible in the cervical and lumbar ventral horn of the spinal cords at 12–13 weeks of age (
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.
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.
Evans Blue, the tracer used to assess disruption of the BSCB, was injected into G93A mice at initial (13 weeks of age) and late (17–18 weeks of age) stages of disease symptoms prior to euthanasia. C57BL/6J mice were also administered the EB dye. In the cervical spinal cord, EB was clearly detected within the blood vessels in the control C57BL/6J mice at 12–13 weeks of age (
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.
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.
Immunofluorescent staining for laminin-1 (major non-collagenous basement membrane glycoprotein) demonstrated well organized microvasculature networks in the cervical and lumbar ventral horn of the spinal cords in C57BL/6J mice at 12–13 weeks of age (
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.
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.
Immunofluorescent staining for Glut-1 (glucose transporter 1) showed high expression of Glut-1 in microvascular endothelia of the cervical and lumbar ventral horn of the spinal cords in C57BL/6J mice at 12–13 weeks of age (
Immunohistochemically, endothelial cells (CD146) and astrocytes (GFAP) were of normal appearance in the cervical and lumbar ventral horn of the spinal cords in the control C57BL/6J mice at 12–13 weeks of age (data not shown) and 19–20 weeks of age (
(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.
(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.
In the present study, we investigated the functional competence of the BSCB in G93A SOD1 mice modeling ALS at different stages of disease. We observed microscopic evidence of BSCB impairment in cervical and lumbar spinal cords, areas of motor neuron degeneration, of ALS mice at initial disease symptoms and, more severely, at late stage disease. Our data show EB leakage in cervical/lumbar spinal cord microvessels in G93A mice at early (13 weeks of age) and late (17–18 weeks of age) stage disease. More leakage was found in lumbar spinal cords of mice at terminal stage disease. Additionally, basement membrane disruption was noted at both early and late stage disease, as shown by the loss of laminin staining in the G93A mice. Downregulation of Glut-1 and CD146 expressions in spinal cord endothelial cells was also found in G93A mice at early and late stage disease and may relate to altered endothelial lining leading to vascular leakage. Small numbers of delineated astrocytes were also established. These results confirm our previous ultrastructural findings
Significant death of motor neurons in G93A mice occurs at the onset of clinical disease (90 days) and by end-stage disease (136 days), mice show up to a 50% loss of cervical and lumbar motor neurons
The primary BBB/BSCB function is control of the CNS homeostasis by selective transport of molecules and cells from the systemic compartment. Substances with a molecular weight higher than 400 Da generally cannot cross the barriers by free diffusion. However, certain endogenous large molecules, such as insulin, leptin, transferrin, and insulin-like growth factors, enter the brain from blood via specific endothelial carrier-mediated or receptor-mediated transporters (reviewed in
Our finding of Evans blue extravasion in early symptomatic G93A mice may suggest that large molecules such as IgG and other blood proteins appear in the spinal cord due to vascular leakage, one possible mechanism accelerating motor neuron damage. However, it is unclear if BBB/BSCB disruption appears prior to motor neuron degeneration or as result of motor neuron dysfunction. Also, differences between the BBB and BSCB in endothelial protein concentrations may impact observed pathological changes in G93A mice. It has been shown that microvascular endothelial cells, isolated from murine spinal cord, morphologically similar to BBB endothelial cells, express reduced amounts of several prominent BBB proteins such as tight junction-associated proteins ZO-1 and occluding, adherens junction-associated proteins beta-catenin and VE-cadherin, and the efflux transporter P-glycoprotein
Reduction in immunofluorescent labeling of basement membrane of affected G93A mice suggests possible membrane disruption. The basement membrane is part of the extracellular matrix and is composed of collagens, proteoglycans, elastin and several glycoproteins, of which laminin is the most abundant
It is well known that glucose transport through the BBB (BSCB) is mediated by glucose transporter isoform 1 (Glut-1)
Another of our findings was that endothelia surrounding capillaries were partially revealed by CD146 antigen expression in the cervical and lumbar spinal cords of G93A mice at initial and, more markedly, at late stages of disease. Moreover, small numbers of delineated astrocytes were established. These results may indicate that degeneration or, at least, partial dysfunction, of non-neuronal cells in ALS occurs. Evidence of widespread inflammatory reactions in ALS already exists. The presence of monocyte/macrophage cells, activated microglia, and reactive astrocytes was established in the spinal cord tissue of most ALS patients
Thus, our results confirm our previous ultrastructural findings on disruption of the BSCB showing functional incompetence of BSCB structural elements in ALS mice. A breakdown in the BSCB is clearly indicated by EB leakage in cervical/lumbar spinal cord microvessels in G93A mice at early and late stages of disease. Laminin labeling suggests that basement membrane of vessels in the spinal cords of the diseased G93A mice may be affected. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB may be related to altered endothelial lining leading to vascular leakage. Degeneration of astrocytes could influence BSCB integrity. Importantly, is BSCB breakdown a primary or secondary mechanism to motor neuron degeneration in G93A mice? Demonstrating BSCB disruption prior to the onset of disease symptoms and other pathological processes would indicate that BSCB disruption plays a primary role in ALS pathogenesis.
All described procedures were approved by the Institutional Animal Care and Use Committee at USF and conducted in compliance with the
Body weight was measured weekly throughout the study. Extension reflex was also observed weekly, as we previously described
Evans Blue dye (EB, Aldrich Chemical), 961 Da, was used as a tracer for assessing BBB disruption
Euthanatasia of all mice was achieved under deep pentobarbital anesthesia and perfusion was not performed to avoid mechanical disruption of blood capillaries. The cervical/lumbar spinal cords were removed, fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.2, and then cryoprotected in 20% sucrose in 0.1 M PB (pH 7.2) overnight. Coronal sections of the cervical (C2–C3) and lumbar (L4–L5) spinal cords were cut at 30 µm in a cryostat.
For identification of EB leakage, serial tissue sections of the spinal cords were thaw-mounted on slides, washed with deionized water to remove the freezing medium, and then rinsed several times in phosphate-buffered saline (PBS). The slides were coverslipped with Vectashield (Vector) and examined under an epifluorescence microscope.
Some spinal cord tissues were used for immunofluorescent analysis of the basement membrane (laminin), endothelial cells (Glut-1, CD146), and astrocytes (GFAP). Briefly, sections of the cervical/lumbar spinal cords were labeled with rat anti-mouse laminin-1 (α and β chains) monoclonal antibody (1∶50, Chemicon), rabbit anti-mouse Glut-1 polyclonal antibody (1∶100, Alpha Diagnostic Int.), or double-stained with mouse monoclonal CD146 (1∶30, Chemicon) and rabbit polyclonal antibodies against glial fibrillary acidic protein (GFAP, 1∶500, Dako). The next day, the slides were incubated for 2 hrs with appropriate secondary antibodies conjugated to either rhodamine (1∶1500, Alexa 594, Molecular Probes) or FITC (1∶500–700, Alexa 488, Molecular Probes) and, after several rinses in PBS, coverslipped with Vectashield or Vectashield with DAPI (Vector) and examined under epifluorescence using an Olympus BX60 microscope.
Coronal sections of the C2–C3 and L4–L5 spinal cords were rinsed in PBS and then stained with 0.1% cresyl violet (30 sec) for routine histological analysis of motor neurons. Sections were then washed several times in deionized water, air-dried, dehydrated, and coverslipped using Permount.
Data are presented as means±SEM. The nonparametric Mann-Whitney unpaired test was used to compare medians.