muramidase and Spinal-Cord-Injuries

The role of neuronal Toll-like receptor 4 (TLR4) in nerve injury is being pursued actively. However, the endogenous activation of neuronal TLR4 during neuroinflammation, in absence of the participation of glial TLR4, remains elusive. Here, we identified lysozyme as an endogenous activator of neuronal TLR4 signaling during nerve injury. Upon nerve injury, enhanced expression of lysozyme promoted neuronal hyperexcitability and neuropathic pain. Injections of lysozyme in healthy rats increased their mechanical and thermal pain sensitivity. Likewise, infusion of spinal cord slices with lysozyme increased neuronal excitability typical of neuropathic pain. Our results also showed that lysozyme activated excitability of both Aδ- and C-fibers. Thus, in addition to the discovery of lysozyme as an endogenous ligand for regulating neuronal TLR4 signaling, this study also lays the foundation of our understanding of its role in nervous system pathologies, providing multiple avenues for treating neuroinflammation.

Topics: Animals; Annexin A2; Cell Membrane; Disease Models, Animal; Female; Ganglia, Spinal; Humans; Injections; Kinetics; Muramidase; Nerve Tissue; Neuralgia; Neurons; Nociceptors; Protein Binding; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Toll-Like Receptor 4; Up-Regulation

Resident microglia and infiltrating myeloid cells play important roles in the onset, propagation, and resolution of inflammation in central nervous system (CNS) injury and disease. Identifying cell type-specific mechanisms will help to appropriately target interventions for tissue repair. Arginase-1 (Arg-1) is a well characterised modulator of tissue repair and its expression correlates with recovery after CNS injury. Here we assessed the cellular localisation of Arg-1 in two models of CNS damage. Using microglia specific antibodies, P2ry12 and Fc receptor-like S (FCRLS), we show the LysM-EGFP reporter mouse is an excellent model to distinguish infiltrating myeloid cells from resident microglia. We show that Arg-1 is expressed exclusively in infiltrating myeloid cells but not microglia in models of spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). Our in vitro studies suggest that factors in the CNS environment prevent expression of Arg-1 in microglia in vivo. This work suggests different functional roles for these cells in CNS injury and repair and shows that such repair pathways can be switched on in infiltrating myeloid cells in pro-inflammatory environments.

Topics: Animals; Arginase; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Green Fluorescent Proteins; Inflammation; Mice; Mice, Inbred C57BL; Microglia; Muramidase; Myeloid Cells; Spinal Cord Injuries

The role of hematogenous (hMΦ) and microglial (mMΦ) macrophages following spinal cord injury (SCI) remains unclear as they are not distinguished easily from each other in the lesion area. We have recently described the temporal and spatial response to SCI of each MΦ population using the lys-EGFP-ki mouse that enables EGFP(+) hMΦ to be distinguished from EGFP(-) mMΦ at the lesion site. In the present study, we characterized the response of monocyte and hMΦ subsets and mMΦ to SCI. We describe, for the first time, the responses of circulating classical (pro-inflammatory) and non-classical monocyte subsets to SCI. Additionally, we show the presence of classical and non-classical hMΦ at the SCI lesion. Importantly, we demonstrate that the 'classical pro-inflammatory' hMΦ respond in the acute (1d, 3d) stages of SCI while the 'non-classical' hMΦ respond in the sub-acute (7d, 14d) phase of SCI. At later time points (6weeks post injury) classical hMΦ return to the injury site. Our study offers new insight into the cellular inflammatory response that occurs after SCI and suggests that the timing and targets of anti-inflammatory therapies may be crucial to maximize neuroprotection at the acute and more chronic stages of SCI.

Topics: Animals; Disease Models, Animal; Female; Flow Cytometry; Green Fluorescent Proteins; Macrophages; Male; Mice; Mice, Transgenic; Microglia; Monocytes; Muramidase; Spinal Cord Injuries; Time Factors

After spinal cord injury (SCI), resident and peripheral myelomonocytic cells are recruited to the injury site and play a role in injury progression. These cells are important for clearing cellular debris, and can modulate the retraction and growth of axons in vitro. However, their precise spatiotemporal recruitment dynamics is unknown, and their respective roles after SCI remain heavily debated. Using chronic, quantitative intravital two-photon microscopy of adult mice with SCI, here we show that infiltrating lysozyme M (LysM(+)) and resident CD11c(+) myelomonocytic cells have distinct spatiotemporal recruitment profiles, and exhibit changes in morphology, motility, phagocytic activity and axon interaction patterns over time. This study provides the first in vivo description of the influx of inflammatory and resident myelomonocytic cells into the injured spinal cord and their interactions with cut axons, and underscores the importance of precise timing and targeting of specific cell populations in developing therapies for SCI.

Topics: Animals; Axons; CD11c Antigen; Cell Movement; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence, Multiphoton; Monocytes; Muramidase; Optical Imaging; Phagocytosis; Spinal Cord Injuries

PLGA particles have been extensively used as a sustained drug-delivery system, but there are multiple drawbacks when delivering proteins. The focus of this work is to address the most significant disadvantages to the W/O/W double emulsion procedure and demonstrate that simple changes to this procedure can have significant changes to particle size and dispersity and considerable improvements to protein loading, activity and sustained active protein release. A systematic approach was taken to analyze the effects of the following variables: solvent miscibility (dichloromethane (DCM), ethyl acetate, acetone), homogenization speed (10 000-25 000 rpm), PLGA concentration (10-30 mg/ml) and additives in both the organic (sucrose acetate isobutyrate (SAIB)) and aqueous (bovine serum albumin (BSA)) phases. Increasing solvent miscibility decreased particle size, dispersity and protein denaturation, while maintaining adequate protein loading. Increasing solvent miscibility also lowered the impact of homogenization on particle size and dispersity and protein activity. Changes to PLGA concentration demonstrated a minimum impact on particle size and dispersity, but showed an inverse relationship between protein encapsulation efficiency and particle protein weight percent. Most particles tested provided sustained release of active protein over 60 days. Increasing solvent miscibility resulted in increases in the percent of active protein released. When subjected to synthesis conditions with DCM as the solvent, BSA as a stabilizer resulted in the maximum stabilization of protein at a concentration of 100 mg/ml. At this concentration, BSA allowed for increases in the total amount of active protein delivered for all three solvents. The benefit of SAIB was primarily increased protein loading.

Topics: Acetates; Acetone; Animals; Cattle; Delayed-Action Preparations; Lactic Acid; Methylene Chloride; Muramidase; Nanoparticles; Nerve Regeneration; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Protein Denaturation; Serum Albumin, Bovine; Solubility; Solvents; Spinal Cord Injuries; Sucrose

The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined.

Topics: Animals; Antigens, CD1; Antigens, Differentiation; Antigens, Ly; Clodronic Acid; Disease Models, Animal; Female; Flow Cytometry; Gene Expression Regulation; Green Fluorescent Proteins; Liposomes; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Muramidase; Neutrophils; Peroxidase; Spinal Cord Injuries; Time Factors

The evolution of tissue damage in compressive spinal cord injuries in rats was studied using an immunohistochemical technique and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The rupture of small vessels accompanied by intense tissue permeation of serum components in and around the hemorrhagic foci appeared to be immediate consequences of the mechanical insult. The loss of cell membrane integrity in neural elements became evident within 1 hour after injury as shown by the diffuse albumin-immunoreactivity of the cytoplasm. At the site of mechanical insult, approximately 30% of the neurofilament proteins were degraded within 1 hour, and 70% of them were lost within 4 hours after injury. A large number of cells positive for glial fibrillary acidic protein were found to demarcate the injured tissue within 1 hour after injury. The progression of tissue damage largely subsided within 48 hours. One week after injury, severe degeneration of the ascending tracts in the posterior funiculus was shown clearly by axon staining and less convincingly by myelin staining. Secondary degeneration of the corticospinal tract in distal segments remained inconspicuous for up to 3 months.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Glial Fibrillary Acidic Protein; Intermediate Filament Proteins; Male; Muramidase; Neurofilament Proteins; Rats; Spinal Cord; Spinal Cord Injuries; Time Factors

Topics: Bacteriuria; Follow-Up Studies; Humans; Kidney Calculi; Muramidase; Paraplegia; Proteinuria; Pyelonephritis; Pyuria; Spinal Cord Injuries