Live imaging of platelets and neutrophils during antibody-mediated neurovascular thrombosis.

Immune complexes form in systemic disorders such as rheumatological, autoimmune, and allergic diseases or in response to infections or medications. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccines have been associated with rare yet serious thrombotic complications in the brain due to the formation of immune complexes that activate platelets. There are currently no data visualizing the interplay of platelets with leukocytes and the brain vasculature endothelium in response to immune complexes. This is in part due to the absence of FcγRIIA in mice, a receptor for immune complexes implicated in these thrombotic incidents. Here, we describe and illustrate events at the cellular level that take place in the brain vasculature in response to systemic administration of surrogate immune complexes. We used Ly6gCre+/-::Rosa26-TdT+/-::CD41-YFP+/- mice expressing the FcγRIIA transgene and fluorescence in neutrophils and platelets. Using real-time videomicroscopy to capture high-velocity events in conjunction with unbiased computer-assisted analyses, we provide images and quantifications of the cellular responses downstream of FcγRIIA stimulation. We observed transient and stable platelet-neutrophil interactions, platelets forming thrombi, and neutrophil adhesion to blood vessel walls. This imaging approach in a quadruple transgenic animal model can be used for the study of the pathogenic roles of immune complexes in disease.

Requirement of myeloid cells for axon regeneration.

The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TK(mt-30) mice expressing a mutated HSV-1 thymidine kinase (TK) gene regulated by the myeloid-specific CD11b promoter. Continuous delivery of ganciclovir at a sciatic nerve lesion site greatly decreased the number of granulocytes/inflammatory monocytes and macrophages in the distal stump of CD11b-TK(mt-30) mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TK(mt-30) mice. This was caused by an unsuitable growth environment rather than an altered regeneration capacity of neurons. In absence of CD11b+ cells, the clearance of inhibitory myelin debris was prevented, neurotrophin synthesis was abolished, and blood vessel formation/maintenance was severely compromised in the sciatic nerve distal stump. Spinal cord-injured axons also failed to regenerate through peripheral nerve grafts in the absence of CD11b+ cells. Therefore, myeloid cells support axonal regeneration and functional recovery by creating a growth-permissive milieu for injured axons.

Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury.

Toll-like receptors (TLRs) bind specific components conserved among microorganisms as well as endogenous ligands produced by necrotic cells, injured axons, and the extracellular matrix. Here, we investigated whether TLRs are involved in regulating the immune response, Wallerian degeneration (WD), and nerve regeneration after sciatic nerve lesion. Early expression of interleukin-1beta and monocyte chemoattractant protein-1 was compromised in the sciatic nerve distal stump of mice deficient in TLR signaling. In addition, significantly fewer macrophages were recruited and/or activated in the sciatic nerve distal stump of TLR2-, TLR4-, and MyD88-deficient mice compared with wild-type littermates, whereas WD, axonal regeneration, and recovery of locomotor function were impaired. In contrast, animals that received a single microinjection of TLR2 and TLR4 ligands at the site of sciatic nerve lesion had faster clearance of the degenerating myelin and recovered earlier than saline-injected control rats. Finally, rats that had altered innate immune response through dexamethasone treatment exhibited three times more myelin debris in their sciatic nerve distal stump and a significant delay in recovery of locomotor function. Our results provide strong evidence that TLR signaling plays a critical role in orchestrating the innate immune response leading to efficient and rapid clearance of inhibitory myelin debris and nerve regeneration.

Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord.

The phagocytic cell response within the injured spinal cord is inefficient, allowing myelin debris to remain for prolonged periods of time within white matter tracts distal to the injury. Several proteins associated with this degenerating myelin are inhibitory to axon growth and therefore prevent severed axons from regenerating. Inflammatory agents such as lipopolysaccharide (LPS) can stimulate both the migration and phagocytic activity of macrophages. Using in situ hybridization, we found that the expression of the LPS membrane receptor, CD14, was enhanced in the mouse dorsal column following a dorsal hemisection. Double labeling studies showed that microglia and macrophages are the two major cell types expressing CD14 mRNA following spinal cord injury (SCI). We therefore tested whether systemic injections of LPS would increase the number and phagocytic activity of macrophages/microglia in the ascending sensory tract (AST) of the mouse dorsal column following a dorsal hemisection. Mice were treated daily via intraperitoneal injections of either LPS or phosphate-buffered saline (PBS). At 7 days post-SCI, greater numbers of activated mononuclear phagocytes were present in the AST undergoing Wallerian degeneration (WD) in LPS-treated animals compared with controls. Animals treated with LPS also exhibited greater Oil Red O staining, which is specific for degenerating myelin and macrophages phagocytosing myelin debris. Myelin clearance was confirmed at 7 days using Luxol Fast Blue staining and on toluidine blue-stained semi-thin sections. These results indicate that it is possible to manipulate the innate immune response to accelerate myelin clearance during WD in the injured mouse spinal cord.