Supplementary Materials

Supplementary Material for:

Targeted complement inhibition salvages stressed neurons and inhibits neuroinflammation after stroke in mice

Ali Alawieh, E. Farris Langley, Stephen Tomlinson*

*Corresponding author. Email: tomlinss{at}musc.edu

Published 16 May 2018, Sci. Transl. Med. 10, eaao6459 (2018)
DOI: 10.1126/scitranslmed.aao6459

This PDF file includes:

  • Materials and Methods
  • Fig. S1. B4Crry inhibits complement activation and IgM deposition on stressed neurons and endothelial cells in vitro.
  • Fig. S2. B4Crry is retained specifically in target tissue with no effects on systemic complement activity.
  • Fig. S3. B4Crry localizes to both the cerebral vasculature and parenchyma after murine stroke.
  • Fig. S4. B4Crry inhibits acute and subacute IgM and C3d deposition in the penumbra.
  • Fig. S5. B4Crry does not provide additional neuroprotection in C3−/− mice.
  • Fig. S6. Three-dimensional reconstruction of lesions using Nissl-stained images.
  • Fig. S7. B4Crry reduces lesion expansion 15 days after MCAO.
  • Fig. S8. CR2Crry does not provide sustained neuroprotection beyond the acute phase of MCAO.
  • Fig. S9. B4Crry does not increase mortality in a murine pneumonia model.
  • Fig. S10. Live perilesional neurons express c-fos, and microglial cells are attracted to c-fos+ neurons.
  • Fig. S11. Inhibition of complement by B4Crry inhibits microglial phagocytosis of stressed penumbra neurons.
  • Fig. S12. B4Crry administered at 6 hours after MCAO also inhibits microglial phagocytosis of c-fos+ material.
  • Fig. S13. B4Crry modulates complement-neuronal interaction.
  • Fig. S14. B4Crry treatment preserves homeostatic microglial activity after stroke.
  • Fig. S15. B4Crry affects immune system–related gene expression 5 days after MCAO.
  • Fig. S16. Acute targeted inhibition of complement prevents chronic amplification of microglial activation weeks after MCAO.
  • Fig. S17. Protocol for quantification of neuronal, dendritic, and microglial density.
  • Fig. S18. Microgliosis is associated with loss of neurons after stroke.
  • Fig. S19. B4Crry inhibits chronic IgM and C3d deposition in the brain 15 days after MCAO.
  • Fig. S20. Modified annexin IV DAMP is expressed in postmortem human ischemic brain as assessed by B4scFv immunostaining.
  • Fig. S21. B4 IgM and B4Crry bind to the ischemic penumbra of acute stroke patients.
  • Fig. S22. Overview of the mechanism of action of B4Crry and the experimental paradigm.
  • Fig. S23. Complement inhibition or deficiency did not affect regional cerebral blood flow after MCAO and reperfusion.
  • Table S1. Characteristics of human stroke brain donors from which samples were used in immunostaining.
  • Table S2. Detailed description of antibodies used in immunostaining studies.
  • Legends for movies S1 to S7
  • References (3239)

[Download PDF]

Other Supplementary Material for this manuscript includes the following:

  • Movie S1 (.mp4 format). Representative field of c-fos material within microglia of vehicle-treated controls.
  • Movie S2 (.mp4 format). Representative field of c-fos material within microglia of B4scFv-treated mice.
  • Movie S3 (.mp4 format). Representative field of c-fos material within microglia of B4Crry-treated mice.
  • Movie S4 (.mp4 format). Representative field of c-fos material within microglia of C3-deficient mice.
  • Movie S5 (.mp4 format). Representative field showing attraction of Iba1+ microglia to c-fos+/NeuN+ neurons that are tagged by C3d opsonins.
  • Movie S6 (.mp4 format). Representative field showing neuronal material (NeuN, red) within Lamp1+ vesicles inside Mac2+ inflammatory microglia.
  • Movie S7 (.mp4 format). Representative field showing c-fos material (c-fos, red) within Lamp1+ vesicles inside Iba1+ microglia.