Blood-brain barrier resealing in neuromyelitis optica occurs independently of astrocyte regeneration
Anne Winkler, Claudia Wrzos, Michael Haberl, Marie-Theres Weil, Ming Gao, Wiebke Möbius, Francesca Odoardi, Dietmar R. Thal, Mayland Chang, Ghislain Opdenakker, Jeffrey L. Bennett, Stefan Nessler, Christine Stadelmann
Anne Winkler, Claudia Wrzos, Michael Haberl, Marie-Theres Weil, Ming Gao, Wiebke Möbius, Francesca Odoardi, Dietmar R. Thal, Mayland Chang, Ghislain Opdenakker, Jeffrey L. Bennett, Stefan Nessler, Christine Stadelmann
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Research Article Neuroscience

Blood-brain barrier resealing in neuromyelitis optica occurs independently of astrocyte regeneration

Abstract

Approximately 80% of neuromyelitis optica spectrum disorder (NMOSD) patients harbor serum anti–aquaporin-4 autoantibodies targeting astrocytes in the CNS. Crucial for NMOSD lesion initiation is disruption of the blood-brain barrier (BBB), which allows the entrance of Abs and serum complement into the CNS and which is a target for new NMOSD therapies. Astrocytes have important functions in BBB maintenance; however, the influence of their loss and the role of immune cell infiltration on BBB permeability in NMOSD have not yet been investigated. Using an experimental model of targeted NMOSD lesions in rats, we demonstrate that astrocyte destruction coincides with a transient disruption of the BBB and a selective loss of occludin from tight junctions. It is noteworthy that BBB integrity is reestablished before astrocytes repopulate. Rather than persistent astrocyte loss, polymorphonuclear leukocytes (PMNs) are the main mediators of BBB disruption, and their depletion preserves BBB integrity and prevents astrocyte loss. Inhibition of PMN chemoattraction, activation, and proteolytic function reduces lesion size. In summary, our data support a crucial role for PMNs in BBB disruption and NMOSD lesion development, rendering their recruitment and activation promising therapeutic targets.

Authors

Anne Winkler, Claudia Wrzos, Michael Haberl, Marie-Theres Weil, Ming Gao, Wiebke Möbius, Francesca Odoardi, Dietmar R. Thal, Mayland Chang, Ghislain Opdenakker, Jeffrey L. Bennett, Stefan Nessler, Christine Stadelmann

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Figure 1

Early loss of astrocytes coincides with BBB breakdown in experimental NMOSD lesions.

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Early loss of astrocytes coincides with BBB breakdown in experimental NM...
To assess the time course of astrocyte lesion development, AQP4 Abs and human complement were injected intracortically and animals were perfused after 3 hours, 6 hours, 24 hours, 3 days, and 7 days. Controls were injected with an irrelevant Ab in the presence of human complement. Three hours after Ab and complement injection, GFAP-positive astrocytes were still observed at the injection site (A). Monastral blue marks the injection site. However, dying GFAP-positive cells with retracting processes were also found (A, insert). Twenty-four hours after injection, large, well-demarcated areas with loss of GFAP (B) and AQP4 immunoreactivity (C; serial sections of the same lesion, astrocyte loss marked by dotted line) were detected. Quantification of GFAP immunoreactivity revealed initial loss 6 hours after lesion induction, peaking between 24 hours and 3 days, with a subsequent repopulation of GFAP-positive cells. No astrocyte loss is observed after injection of control Ab (ctrl-Ab) together with human complement. Number of lesions: 3 hours, n = 3; 6 hours and 24 hours, n = 10; 3 days, AQP4/2B4, n = 6/4; 7 days, n = 5 (D). Simultaneously, with the loss of astrocytes, a prominent extravasation of the injected tracers FITC-albumin (60 kDa) (E) and Texas Red cadaverine (0,69 kDa) (F) into the brain parenchyma was observed 6 hours after focal injection. No vascular leakage of either molecule was detected 24 hours after stereotactic injection (G, FITC-albumin; H, Texas Red cadaverine), which is confirmed by quantification (I). Number of lesions: FITC-albumin: 3 hours, n = 3; 6 hours, n = 8; 24 hours, n = 9; 3 days, n = 4. (J) Texas Red cadaverine, n = 3. (D, I, and J) Kruskal-Wallis test followed by Dunn’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001. Graphs are shown as mean ± SEM. Scale bars: 100 μm (A–C); 500 μm (E–H).