Dataset Information

Intravital imaging of axonal interactions with microglia and macrophages in a mouse dorsal column crush injury.

ABSTRACT: Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

SUBMITTER: Evans TA

PROVIDER: S-EPMC4275021 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Intravital imaging of axonal interactions with microglia and macrophages in a mouse dorsal column crush injury.

Evans Teresa A TA Barkauskas Deborah S DS Myers Jay T JT Huang Alex Y AY

Journal of visualized experiments : JoVE 20141123 93

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain an ...[more]

PMID: 25489963

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Project description:After traumatic spinal cord injury, functional deficits increase as axons die back from the center of the lesion and the glial scar forms. Axonal dieback occurs in two phases: an initial axon intrinsic stage that occurs over the first several hours and a secondary phase which takes place over the first few weeks after injury. Here, we examine the secondary phase, which is marked by infiltration of macrophages. Using powerful time-lapse multi-photon imaging, we captured images of interactions between Cx3cr1(+/GFP) macrophages and microglia and Thy-1(YFP) axons in a mouse dorsal column crush spinal cord injury model. Over the first few weeks after injury, axonal retraction bulbs within the lesion are static except when axonal fragments are lost by a blebbing mechanism in response to physical contact followed by phagocytosis by mobile Cx3Cr1(+/GFP) cells. Utilizing a radiation chimera model to distinguish marrow-derived cells from radio-resistant CNS-resident microglia, we determined that the vast majority of accumulated cells in the lesion are derived from the blood and only these are associated with axonal damage. Interestingly, CNS-resident Cx3Cr1(+/GFP) microglia did not increasingly accumulate nor participate in neuronal destruction in the lesion during this time period. Additionally, we found that the blood-derived cells consisted mainly of singly labeled Ccr2(+/RFP) macrophages, singly labeled Cx3Cr1(+/GFP) macrophages and a small population of double-labeled cells. Since all axon destructive events were seen in contact with a Cx3Cr1(+/GFP) cell, we infer that the CCR2 single positive subset is likely not robustly involved in axonal dieback. Finally, in our model, deletion of CCR2, a chemokine receptor, did not alter the position of axons after dieback. Understanding the in vivo cellular interactions involved in secondary axonal injury may lead to clinical treatment candidates involving modulation of destructive infiltrating blood monocytes.

Project description:BACKGROUND:Microglia/macrophages (M/Ms) with multiple functions derived from distinct activation states are key surveillants maintaining brain homeostasis. However, their activation status and role during the brain metastasis of malignant tumors have been poorly characterized. METHODS:Heterozygous CX3CR1-GFP transgenic mice were used to visualize the dynamic changes of M/Ms during the development of experimental brain metastasis through long-term intravital imaging equipped with redesigned bilateral cranial windows. The occurrence of experimental brain metastasis was evaluated after M/Ms were depleted with PLX3397, a CSF-1R inhibitor. The possible mediators of M/Ms in facilitating the brain metastasis were determined using reverse transcription-PCR, immunofluorescence, correlational analysis, and MMP inhibition. RESULTS:Here, we showed that M/Ms were persistently activated and facilitated the formation of melanoma brain metastasis in vivo. We observed that M/Ms gradually and massively accumulated in the metastasis, with a 2.89-fold increase. To precisely depict the dynamic changes in the activation state of M/Ms, we defined the branching parameter to quantify their morphological alterations. The quantitative data showed that the extent of activation of M/Ms in metastatic foci was enhanced, with a 2.27-fold increase from day 1 to day 21. Along with the activation, the M/Ms increased their moving velocity (4.15-fold) and established a rapid, confined, and discontinuous motility behavior. The occurrence of melanoma brain metastasis was significantly hindered under M/M elimination, indicating the key role of M/Ms in the experimental brain metastasis. Interestingly, we found that M/Ms highly expressed matrix metalloproteinase 3 (MMP3), which were strongly correlated with M/M activation and the decrease of tight junction protein zonula occludens-1 (ZO-1). An MMP inhibitor moderately decreased the occurrence of melanoma brain metastasis, suggesting that MMP3 secreted by M/Ms may facilitate melanoma cell growth. CONCLUSIONS:Our results indicated that the activated M/Ms were essential in the development of melanoma brain metastasis, suggesting that M/Ms are a potential therapeutic target for tumor brain metastasis.

Project description:Background: Fibroblast growth factor receptors (FGFRs) are key targets for nerve regeneration and repair. The therapeutic effect of exogenous recombinant FGFs in vivo is limited due to their high molecular weight. Small peptides with low molecular weight, easy diffusion, low immunogenicity, and nontoxic metabolite formation are potential candidates. The present study aimed to develop a novel low-molecular-weight peptide agonist of FGFR to promote nerve injury repair. Methods: Phage display technology was employed to screen peptide ligands targeting FGFR2. The peptide ligand affinity for FGFRs was detected by isothermal titration calorimetry. Structural biology-based computer virtual analysis was used to characterize the interaction between the peptide ligand and FGFR2. The peptide ligand effect on axon growth, regeneration, and behavioral recovery of sensory neurons was determined in the primary culture of sensory neurons and dorsal root ganglia (DRG) explants in vitro and a rat spinal dorsal root injury (DRI) model in vivo. The peptide ligand binding to other membrane receptors was characterized by surface plasmon resonance (SPR) and liquid chromatography-mass spectrometry (LC-MS)/MS. Intracellular signaling pathways primarily affected by the peptide ligand were characterized by phosphoproteomics, and related pathways were verified using specific inhibitors. Results: We identified a novel FGFR-targeting small peptide, CH02, with seven amino acid residues. CH02 activated FGFR signaling through high-affinity binding with the extracellular segment of FGFRs and also had an affinity for several receptor tyrosine kinase (RTK) family members, including VEGFR2. In sensory neurons cultured in vitro, CH02 maintained the survival of neurons and promoted axon growth. Simultaneously, CH02 robustly enhanced nerve regeneration and sensory-motor behavioral recovery after DRI in rats. CH02-induced activation of FGFR signaling promoted nerve regeneration primarily via AKT and ERK signaling downstream of FGFRs. Activation of mTOR downstream of AKT signaling augmented axon growth potential in response to CH02. Conclusion: Our study revealed the significant therapeutic effect of CH02 on strengthening nerve regeneration and suggested a strategy for treating peripheral and central nervous system injuries.

Dataset Information

Intravital imaging of axonal interactions with microglia and macrophages in a mouse dorsal column crush injury.

Publications

Intravital imaging of axonal interactions with microglia and macrophages in a mouse dorsal column crush injury.

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