Ligation of the jugular veins does not result in brain inflammation or demyelination in mice

Abstract

An alternative hypothesis has been proposed implicating chronic cerebrospinal venous insufficiency (CCSVI) as a potential cause of multiple sclerosis (MS). We aimed to evaluate the validity of this hypothesis in a controlled animal model. Animal experiments were approved by the institutional animal care committee. The jugular veins in SJL mice were ligated bilaterally (n = 20), and the mice were observed for up to six months after ligation. Sham-operated mice (n = 15) and mice induced with experimental autoimmune encephalomyelitis (n = 8) were used as negative and positive controls, respectively. The animals were evaluated using CT venography and (99m)Tc-exametazime to assess for structural and hemodynamic changes. Imaging was performed to evaluate for signs of blood-brain barrier (BBB) breakdown and neuroinflammation. Flow cytometry and histopathology were performed to assess inflammatory cell populations and demyelination. There were both structural changes (stenosis, collaterals) in the jugular venous drainage and hemodynamic disturbances in the brain on Tc99m-exametazime scintigraphy (p = 0.024). In the JVL mice, gadolinium MRI and immunofluorescence imaging for barrier molecules did not reveal evidence of BBB breakdown (p = 0.58). Myeloperoxidase, matrix metalloproteinase, and protease molecular imaging did not reveal signs of increased neuroinflammation (all p>0.05). Flow cytometry and histopathology also did not reveal increase in inflammatory cell infiltration or population shifts. No evidence of demyelination was found, and the mice remained without clinical signs. Despite the structural and hemodynamic changes, we did not identify changes in the BBB permeability, neuroinflammation, demyelination, or clinical signs in the JVL group compared to the sham group. Therefore, our murine model does not support CCSVI as a cause of demyelinating diseases such as multiple sclerosis.

Figure 1. CT venogram and 3D vessel imaging of JV stenosis.

(A) Sham mouse with patent JVs (white arrow). JVL mouse confirming ligation of the right and left JV and formation of collateral veins (yellow arrow). (B) Sham mouse exhibits normal venous drainage. (C) Right-side view of JVL mouse exhibits formation of small collateral vessels (yellow arrow) and stenosis due to surgical ligation (black arrow). Left-side view of JVL mouse exhibits complete occlusion at ligation site (black arrow) with formation of collateral vessels (yellow arrow).

Figure 2. MR imaging to assess the blood-brain barrier and myeloperoxidase (MPO) activity.

(A) MR imaging with DTPA-Gd does not reveal changes in blood-brain barrier permeability. Sham group (n = 3, CNR (contrast-to-noise ratio) = 0.32±0.17, JVL group (n = 6, CNR = 0.21±0.051, p = 0.58). EAE data provided for comparison (n = 3, 4.7±1.3, p = 0.024 vs. JVL). (B) MPO activity map from MPO-Gd MRI to detect areas of inflammation. A representative sham mouse (left) demonstrates no evidence of MPO activity. A representative JVL mouse (middle) demonstrates no evidence of MPO activity. A stage 2 EAE mouse (right) for comparison.

Figure 3. FMT-CT imaging of MMP and protease activity.

(A) Representative images from sham and JVL mice show no visible signal from FMT imaging. (B) Quantification of the fluorescence signal demonstrates barely measurable signal and no significant difference between sham and JVL mice.

Figure 4. Flow cytometric analysis of brain inflammatory cells.

(A) Cells were pre-gated for positive CD45 expression to identify all leukocytes, and (B) then divided into lymphocytes, neutrophils, and myeloid cells. The total number of all leukocytes, but also lymphocytes, neutrophils, and myeloid cells in the brain was unaffected in JVL mice (n = 4) compared to sham (n = 4), but significantly increased in EAE mice (n = 5). (C) Differentiating macrophages/microglia from monocytes showed that there were almost no monocytes in the brain of JVL and sham mice, but both cell types were increased to high numbers in EAE mice. Lin = CD90, NK1.1, B220, CD49b, and Ly-6G.

Figure 5. Histopathology of sham (top row), JVL, (middle row), and EAE (bottom row) groups.

(A) MPO stain for MPO-positive cells/myeloid cells, (B) Mac-3 stain for activated macrophages/microglia, and (C) LFB stain for demyelination. Bar = 50 µm.

Figure 6. Weights of sham group (n = 6, open circle) and JVL group (n = 16, solid square) up to day 130 post-surgery.