Abstract
Acute focal ischemia was used to test whether reducing store-operated calcium entry (SOCE) in microglia improves stroke outcomes, employing several imaging modalities to resolve different aspects of stroke pathology. A microglia-specific Stim1/Stim2 conditional knockout was generated using the Cx3cr1-CreER driver. Calcium signaling was imaged in vivo by two-photon imaging of GCaMP5G through a cranial window during KCl-evoked cortical spreading depolarizations (CSDs). Knockout microglia showed 34% reductions in Ca^2+ transient peak and area with unchanged duration, confirming attenuation of calcium release-activated channel (CRAC)-mediated influx.
Ischemic injury was then quantified on a 9.4 T Bruker BioSpec system using rigorously calibrated, multi-contrast MRI protocols: T2-weighted two-dimensional rapid acquisition with relaxation enhancement (TurboRARE) for edema, diffusion-weighted imaging (DWI) with a multi-shot echo-planar imaging (EPI) readout and b ≈ 700 s/mm^2 for apparent diffusion coefficient (ADC) mapping via S(b)=S_0[e^(-bD)], and flow-sensitive alternating inversion recovery (FAIR) arterial spin labeling (ASL) to probe perfusion using multiple inversion times. Lesions were segmented in ITK-SNAP; diffusion damage was further normalized by a contralateral mirror-mask method to yield a within-subject ADC ratio. From a measurement standpoint, these combined contrasts separate complementary imaging properties: TurboRARE isolates T2-prolongation from edema; DWI/ADC quantify restricted water motion independent of T2 shine-through; and ASL reports the flow of labeled blood water.
At 6 hours after distal middle cerebral artery occlusion (dMCAo), T2 and ADC volumes did not differ between genotypes (controls: T2 3.80 ± 1.45 mm^3, ADC 2.23 ± 0.92 mm^3; mutants: T2 3.11 ± 2.33 mm^3, ADC 1.80 ± 1.09 mm^3), consistent with a similar early core smaller than the T2-bright edema. ASL confirmed marked hypoperfusion in the middle cerebral artery territory without a group difference at this time point. By 48 h, the imaging results told a different story: T2-defined infarcts were nearly twice as large in the knockout cohort (controls 5.85 ± 4.11 mm^3 vs. mutants 9.98 ± 5.75 mm^3), and mean ADC values in the infarcted areas decreased by 11% in absolute terms (controls 5.62×10^-4 vs. mutants 5.02×10^-4 mm^2/s). Normalized to the contralateral hemisphere, ADC was reduced to 75% in mutants compared to only 84% in controls, indicating more severe diffusion restriction.
Together, these data reveal that blocking microglial Ca^2+ signals via loss of STIM1/STIM2 does not protect brain tissue during acute stroke. Instead, it allows the ischemic core to expand as perfusion remains poor. The findings argue that microglial CRAC-mediated Ca^2+ influx supports early neuroprotective processes whose loss worsens outcome in this aged mouse stroke model. More broadly, the work illustrates how advancements in high-field multi-contrast MRI offer a quantitative and comprehensive evaluation of outcomes in experimental stroke research, surpassing traditional histological assessment.
