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In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.

Morsch M, Radford R, Lee A, Don EK, Badrock AP, Hall TE, Cole NJ, Chung R - Front Cell Neurosci (2015)

Bottom Line: In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord.This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation.This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

View Article: PubMed Central - PubMed

Affiliation: Motor Neuron Disease Research Group, Faculty of Medicine and Health Sciences, Macquarie University Sydney, NSW, Australia.

ABSTRACT
Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

No MeSH data available.


Related in: MedlinePlus

3D rendering demonstrates dynamic morphological features of activated microglia. Confocal live-imaging alone provides basic morphometric information of microglia phagocytosing an UV-ablated spinal neuron (A). Imaris 3D rendering of this same single microglia revealed the formation of a phagosome-like structure (B), which presumably facilitates engulfment of the neuronal remnants. Supplementary Videos 5, 6 show the time-lapse videos of this process for comparison. Scale bars = 20 μm.
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Figure 7: 3D rendering demonstrates dynamic morphological features of activated microglia. Confocal live-imaging alone provides basic morphometric information of microglia phagocytosing an UV-ablated spinal neuron (A). Imaris 3D rendering of this same single microglia revealed the formation of a phagosome-like structure (B), which presumably facilitates engulfment of the neuronal remnants. Supplementary Videos 5, 6 show the time-lapse videos of this process for comparison. Scale bars = 20 μm.

Mentions: Confocal live-imaging alone provides only limited morphometric information upon phagocytosing microglia. Using three-dimensional rendering of time-lapse responses (Imaris), we found repeatedly that phagocytosing microglial cells extend bulbous-like protrusions tipped with phagocytic cups (Figure 7). For example, we observed a single microglia engulfing the neuronal remnants of an UV-ablated spinal neuron through formation of a phagosome-like structure (10 μm in size; Figure 7B, Supplementary Video 6) that cannot readily be resolved through standard confocal microscopy (Figure 7A; Supplementary Video 5). Importantly, this live-imaging observation is in line with scanning electron microscopy data showing that indeed phagosomes form a tight fitting around apoptotic particles (Krysko et al., 2003, 2006). Collectively, our data strongly demonstrates that microglia (in the zebrafish spinal cord) form bulbous-like phagocytic cups to engulf neuronal remnants, equivalent to the phagocytic behavior of mammalian microglia in the brain. Morphometric rendering analysis of a subset of these microglial cells with Imaris confirmed the speed and distances reported above (Table 1). It furthermore revealed that these microglial cells condensed in size during activation, demonstrated by a ~40% reduction in their average volume and surface area (1189.5 μm3 vs. 703.8 μm3; 1433.3 μm2 vs. 852.6 μm2; Table 1; Figures 6H–K).


In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.

Morsch M, Radford R, Lee A, Don EK, Badrock AP, Hall TE, Cole NJ, Chung R - Front Cell Neurosci (2015)

3D rendering demonstrates dynamic morphological features of activated microglia. Confocal live-imaging alone provides basic morphometric information of microglia phagocytosing an UV-ablated spinal neuron (A). Imaris 3D rendering of this same single microglia revealed the formation of a phagosome-like structure (B), which presumably facilitates engulfment of the neuronal remnants. Supplementary Videos 5, 6 show the time-lapse videos of this process for comparison. Scale bars = 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4553390&req=5

Figure 7: 3D rendering demonstrates dynamic morphological features of activated microglia. Confocal live-imaging alone provides basic morphometric information of microglia phagocytosing an UV-ablated spinal neuron (A). Imaris 3D rendering of this same single microglia revealed the formation of a phagosome-like structure (B), which presumably facilitates engulfment of the neuronal remnants. Supplementary Videos 5, 6 show the time-lapse videos of this process for comparison. Scale bars = 20 μm.
Mentions: Confocal live-imaging alone provides only limited morphometric information upon phagocytosing microglia. Using three-dimensional rendering of time-lapse responses (Imaris), we found repeatedly that phagocytosing microglial cells extend bulbous-like protrusions tipped with phagocytic cups (Figure 7). For example, we observed a single microglia engulfing the neuronal remnants of an UV-ablated spinal neuron through formation of a phagosome-like structure (10 μm in size; Figure 7B, Supplementary Video 6) that cannot readily be resolved through standard confocal microscopy (Figure 7A; Supplementary Video 5). Importantly, this live-imaging observation is in line with scanning electron microscopy data showing that indeed phagosomes form a tight fitting around apoptotic particles (Krysko et al., 2003, 2006). Collectively, our data strongly demonstrates that microglia (in the zebrafish spinal cord) form bulbous-like phagocytic cups to engulf neuronal remnants, equivalent to the phagocytic behavior of mammalian microglia in the brain. Morphometric rendering analysis of a subset of these microglial cells with Imaris confirmed the speed and distances reported above (Table 1). It furthermore revealed that these microglial cells condensed in size during activation, demonstrated by a ~40% reduction in their average volume and surface area (1189.5 μm3 vs. 703.8 μm3; 1433.3 μm2 vs. 852.6 μm2; Table 1; Figures 6H–K).

Bottom Line: In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord.This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation.This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

View Article: PubMed Central - PubMed

Affiliation: Motor Neuron Disease Research Group, Faculty of Medicine and Health Sciences, Macquarie University Sydney, NSW, Australia.

ABSTRACT
Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

No MeSH data available.


Related in: MedlinePlus