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Microglial Depletion with Clodronate Liposomes Increases Proinflammatory Cytokine Levels, Induces Astrocyte Activation, and Damages Blood Vessel Integrity

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Abstract

Investigators are increasingly interested in using microglial depletion to study the role of microglia under pathologic conditions. Liposome-encapsulated clodronate is commonly used to eliminate macrophage populations because it causes functionally irreversible inhibition and apoptosis once phagocytized by macrophages. Recent studies have shown that microglia can be depleted in disease models by injecting clodronate liposomes into the brain parenchyma. However, it is unclear whether intracerebral administration of clodronate liposomes is a practical method of eliminating microglia under physiologic conditions or whether microglial depletion induces damage to other brain cells. In this study, injecting 1 μL of clodronate liposomes (7 μg/μL) into the striatum of mice caused ablation of microglia at 1 day that persisted for 3 days. Microglia reappeared in the boundary regions of microglia elimination after 5 days. Importantly, we observed an increase in proinflammatory cytokine levels and an increase in neural/glial antigen 2 and glial fibrillary acidic protein expression in the perilesional region. In contrast, expression levels of myelin basic protein, microtubule-associated protein 2, and postsynaptic protein-95 decreased in the periphery of regions where microglia were depleted. Moreover, clodronate liposome administration decreased the density and integrity of blood vessels in the perilesional regions. In cultured primary neurons, clodronate liposome exposure also attenuated ATP synthesis. Together, these findings suggest that intracerebral administration of clodronate liposomes into brain parenchyma can deplete microglia, but can also damage other brain cells and blood vessel integrity.

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Acknowledgments

We thank Claire Levine for assistance with manuscript preparation.

Funding

This work was supported by the National Institutes of Health (R01NS102583 and R01NS105894 to J. Wang), the American Heart Association (Grant-in-Aid 17GRNT33660766 to J. Wang; Scientist Development Grant 16SDG30980031 to X. Han; Postdoctoral Fellowship Award 17POST33660191 to X. Lan), and a Stimulating and Advancing ACCM Research (StAAR) Award from the Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University.

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  1. Qian Li

    Present address: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, People’s Republic of China

  2. Qian Li

    Present address: Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, 100069, People’s Republic of China

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  1. Department of Anesthesiology/Critical Care Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD, 21205, USA

    Xiaoning Han, Qian Li, Xi Lan, Leena EL-Mufti, Honglei Ren & Jian Wang

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Supplementary Fig 1

Parenchymal injection of clodronate liposomes (Clo-Lip) effectively depletes microglia. a, b Representative images of brain sections 4 days after injection of 1 μL fluorescent Dil-labeled liposomes (Dil-Lip) into striatum (a) or lateral ventricle (b). Numbers in the images indicate anteroposterior ordinate distance to the bregma. Scale bars: (a) 100 μm, (b) 500 μm. Red: Dil-Lip, green: Cx3cr1-GFP. c C57BL/6 mice were administered 1 μL (7 μg/μL) of Clo-Lip via striatal injection. Body weight did not change significantly after 1 or 3 days. n = 9. Data are presented as mean ± SD. d Dot plot represents propidium iodide (PI) staining of homogenized brain tissues from Cx3cr1-GFP mice. The R1 population in the left panel represents all events. The right panel shows subsets of the R1 population: red, live cells (PI-negative) and blue, dead cells (PI-positive). e Cx3cr1-GFP+ microglia in ipsilateral cortex were analyzed by flow cytometry on days 1, 4, and 7 after Clo-Lip striatal injection. Representative dot plots are shown, and percentages of Cx3cr1-GFP+ population are indicated. Red: Cx3cr1-GFP− population, green: Cx3cr1-GFP+ population. (PNG 702 kb)

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Han, X., Li, Q., Lan, X. et al. Microglial Depletion with Clodronate Liposomes Increases Proinflammatory Cytokine Levels, Induces Astrocyte Activation, and Damages Blood Vessel Integrity. Mol Neurobiol 56, 6184–6196 (2019). https://doi.org/10.1007/s12035-019-1502-9

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