Marion Buckwalter, Postdoctoral Faculty Sponsor
BACKGROUND: Ischemic stroke provokes a neuroinflammatory response and simultaneously promotes release of epinephrine and norepinephrine by the sympathetic nervous system. This increased sympathetic outflow can act on beta2-adrenergic receptors expressed by immune cells such as brain-resident microglia and monocyte-derived macrophages (MDMs), but the effect on post-stroke neuroinflammation is unknown. Thus, we investigated how changes in beta2-adrenergic signaling after stroke onset influence the microglia/MDM stroke response, and the specific importance of microglia/MDM beta2-adrenergic receptors to post-stroke neuroinflammation.METHODS: To investigate the effects of beta2-adrenergic receptor manipulation on post-stroke neuroinflammation, we administered the beta2-adrenergic receptor agonist clenbuterol to mice 3h after the onset of photothrombotic stroke. We immunostained to quantify microglia/MDM numbers and proliferation and to assess morphology and activation 3days later. We assessed stroke outcomes by measuring infarct volume and functional motor recovery and analyzed gene expression levels of neuroinflammatory molecules. Finally, we evaluated changes in cytokine expression and microglia/MDM response in brains of mice with selective knockout of the beta2-adrenergic receptor from microglia and monocyte-lineage cells.RESULTS: We report that clenbuterol treatment after stroke onset causes enlarged microglia/MDMs and impairs their proliferation, resulting in reduced numbers of these cells in the peri-infarct cortex by 1.7-fold at 3days after stroke. These changes in microglia/MDMs were associated with increased infarct volume in clenbuterol-treated animals. In mice that had the beta2-adrenergic receptor specifically knocked out of microglia/MDMs, there was no change in morphology or numbers of these cells after stroke. However, knockdown of beta2-adrenergic receptors in microglia and MDMs resulted in increased expression of TNFalpha and IL-10 in peri-infarct tissue, while stimulation of beta2-adrenergic receptors with clenbuterol had the opposite effect, suppressing TNFalpha and IL-10 expression.CONCLUSIONS: We identified beta2-adrenergic receptor signaling as an important regulator of the neuroimmune response after ischemic stroke. Increased beta2-adrenergic signaling after stroke onset generally suppressed the microglia/MDM response, reducing upregulation of both pro- and anti-inflammatory cytokines, and increasing stroke size. In contrast, diminished beta2-adrenergic signaling in microglia/MDMs augmented both pro- and anti-inflammatory cytokine expression after stroke. The beta2-adrenergic receptor may therefore present a therapeutic target for improving the post-stroke neuroinflammatory and repair process.
View details for DOI 10.1186/s12974-019-1506-4
View details for PubMedID 31138227
View details for Web of Science ID 000473116800129
Neuroinflammation plays a key role in neuronal injury following ischemic stroke. Positron emission tomography (PET) imaging of translocator protein 18 kDa (TSPO) permits longitudinal, non-invasive visualization of neuroinflammation in both pre-clinical and clinical settings. Many TSPO tracers have been developed, however it is unclear which tracer is the most sensitive and accurate for monitoring the in vivo spatiotemporal dynamics of neuroinflammation across applications. Hence, there is a need for head-to-head comparisons of promising TSPO-PET tracers across different disease states. Accordingly, the aim of this study was to directly compare two promising second-generation TSPO tracers; 11C-DPA-713 and 18F-GE-180, for the first time at acute and chronic time-points following ischemic stroke. Methods: Following distal middle cerebral artery occlusion (dMCAO) or sham surgery, mice underwent consecutive PET/CT imaging with 11C-DPA-713 and 18F-GE-180 at 2, 6, and 28 days after stroke. T2-weighted magnetic resonance (MR) images were acquired to enable delineation of ipsilateral (infarct) and contralateral brain regions of interest (ROIs). PET images were analyzed by calculating % injected dose per gram (%ID/g) in MR-guided ROIs. Standardized uptake value ratios were determined using the contralateral thalamus as a pseudo-reference region (SUVTh). Ex vivo autoradiography and immunohistochemistry were performed to verify in vivo findings. Results: Significantly increased tracer uptake was observed in the ipsilateral compared to contralateral ROI (SUVTh, 50-60 min summed data) at acute and chronic time-points using 11C-DPA-713 and 18F-GE-180. Ex vivo autoradiography confirmed in vivo findings demonstrating increased TSPO-tracer uptake in infarcted versus contralateral brain tissue. Importantly, a significant correlation was identified between microglial/macrophage activation (CD68 immunostaining) and 11C-DPA-713-PET signal, that was not evident with 18F-GE-180. No significant correlations were observed between TSPO-PET and activated astrocytes (GFAP immunostaining). Conclusion: Both 11C-DPA-713 and 18F-GE-180-PET enable detection of neuroinflammation at early and chronic time-points following cerebral ischemia in mice. 11C-DPA-713-PET reflects the extent of microglial activation in infarcted dMCAO mouse brain tissue more accurately compared to 18F-GE-180, and appears to be slightly more sensitive. These results highlight the potential of 11C-DPA-713 for tracking microglial activation in vivo after stroke, and warrants further investigation in both pre-clinical and clinical settings.
View details for PubMedID 29976695
B lymphocytes are a key pathological feature of multiple sclerosis (MS), and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to non-invasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here we evaluated (64)Cu-Rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using positron emission tomography (PET). Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of Myelin Oligodendrocyte Glycoprotein fragment1-125 (MOG1-125) emulsified in complete Freund's adjuvant. Control mice received complete Freund's adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19h following (64)Cu-Rituximab administration. Mice were perfused and sacrificed after final PET scan, and radioactivity in dissected tissues was measured with a gamma-counter. CNS tissues from these mice were immunostained to quantify B cells or further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice compared to controls at all evaluated time points (e.g., 1h post-injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 %ID/g, p<0.05). (64)Cu-Rituximab-PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice compared to 1.25±0.08 and 2.24±0.11%ID/g for controls, p<0.05 for all regions except striatum and thalamus at 1h post-injection. Similarly, ex vivo biodistribution results revealed notably higher (64)Cu-Rituximab uptake in brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased (64)Cu-Rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using (64)Cu-Rituximab-PET. Results from these studies warrant further investigation of (64)Cu-Rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell-targeted therapeutics en route to the clinic.
View details for PubMedID 28687602