Abstract
Studies on colony-stimulating factor 1 receptor (CSF-1R) inhibition-induced microglia depletion indicated that inhibitor withdrawal allowed the renewal of the microglia compartment via repopulation and resolved the inflammatory imbalance. Therefore, we investigated for the first time (to our knowledge) the effects of microglia repopulation on inflammation and functional outcomes in an ischemic mouse model using translocator protein (TSPO)-PET/CT and MR imaging, ex vivo characterization, and behavioral tests. Methods: Eight C57BL/6 mice per group underwent a 30-min transient occlusion of the middle cerebral artery. The treatment group received CSF-1R inhibitor in 1,200 ppm PLX5622 chow (Plexxikon Inc.) from days 3 to 7 to induce microglia/macrophage depletion and then went back to a control diet to allow repopulation. The mice underwent T2-weighted MRI on day 1 after ischemia and 18F-labeled N,N-diethyl-2-(2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-α]pyrimidine-3-yl)acetamide (18F-DPA-714) (TSPO) PET/CT on days 7, 14, 21, and 30. The percentage injected tracer dose per milliliter within the infarct, contralateral striatum, and spleen was assessed. Behavioral tests were performed to assess motor function recovery. Brains were harvested on days 14 and 35 after ischemia for ex vivo analyses (immunoreactivity and real-time quantitative polymerase chain reaction) of microglia- and macrophage-related markers. Results: Repopulation significantly increased 18F-DPA-714 uptake within the infarct on days 14 (P < 0.001) and 21 (P = 0.002) after ischemia. On day 14, the ionized calcium binding adaptor molecule 1 (Iba-1)-positive cell population showed significantly higher expression of TSPO, CSF-1R, and CD68, in line with microglia repopulation. Gene expression analyses on day 14 indicated a significant increase in microglia-related markers (csf-1r, aif1, and p2ry12) with repopulation, whereas peripheral cell recruitment-related gene expression decreased (cx3cr1 and ccr2), indicative of peripheral recruitment during CSF-1R inhibition. Similarly, uncorrected spleen uptake was significantly higher on day 7 after ischemia with treatment (P = 0.001) and decreased after drug withdrawal. PLX5622-treated mice walked a longer distance (P < 0.001) and more quickly (P = 0.009), and showed greater forelimb strength (P < 0.001), than control mice on day 14. Conclusion: This study highlighted the potential of 18F-DPA-714 PET/CT imaging to track microglia and macrophage repopulation after short-term CSF-1R inhibition in stroke.
Highlights
Microglia play a major role in the stroke-induced neuroinflammatory response, as part of the early proinflammatory and later restorative processes [1]
We demonstrated that colony-stimulating factor 1 receptor (CSF-1R) inhibition-induced microglia/macrophages depletion could be tracked using 18F-DPA-714 radiotracer targeting the translocator protein (TSPO) [7]: TSPOdependent neuroinflammation was significantly decreased within the first weeks after stroke, long-term CSF-1R inhibition was associated with poor disease outcome
The sample size was determined based on effect size (p = 0.05, power: 1-β = 0.80), mortality rates and previous stroke studies [7,15], where we investigated the therapeutic effect of dietary approaches on brain inflammation assessed by 18F-DPA-714 positron emission tomography (PET) imaging
Summary
Microglia play a major role in the stroke-induced neuroinflammatory response, as part of the early proinflammatory and later restorative processes [1]. Microglia survival and proliferation are dependent upon signalling through the colony-stimulating factor 1 receptor (CSF-1R) [2]. Administration of the CSF-1R inhibitor PLX5622 progressively leads to the almost complete microglia depletion after one week of treatment in wild type mice [3]. Microglial depletion in acute/subacute ischemia (1-3 days) was associated to increased immune cell infiltration and aggravated brain inflammation [4,5]. Inhibitor withdrawal triggers microglia repopulation, indicated by microglia proliferation and increased activity. We demonstrated that CSF-1R inhibition-induced microglia/macrophages depletion could be tracked using 18F-DPA-714 radiotracer targeting the translocator protein (TSPO) [7]: TSPOdependent neuroinflammation was significantly decreased within the first weeks after stroke, long-term CSF-1R inhibition was associated with poor disease outcome. PLX5622 represents an attractive microglia/macrophages-targeting pharmacological tool allowing modulation of the inflammatory environment after stroke.
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