AAV9-CaMKII-GCaMP6m

Ischemic stroke is one of the leading causes of adult disability, morbidity, and death worldwide. After stroke, acute neuronal excitotoxicity leads to many deleterious consequences, one of which is intracellular calcium dysregulation, ultimately leading to cell death. Using the fluorescent calcium indicator GCaMP, researchers tracked neuronal population response in freely moving animals immediately following distal middle cerebral artery occlusion (dMCAO) in both the core infarct and peri-infarct regions. The results showed that calcium excitotoxicity after arterial occlusion can generally be divided into two phases: a transient increase in activity lasting tens of minutes, followed by a longer, slower, and sustained increase in the fluorescent signal. The first phase is primarily thought to represent neuronal hyperexcitability, defining the therapeutic window, while the second phase may represent gradual cell death. Importantly, the study found that the level of intracellular calcium following artery occlusion correlated with the infarct size at 24 h demonstrating a direct connection between excitotoxicity and cell death in our stroke model. Furthermore, administration of the NMDA antagonist MK-801 resulted in a reduction in calcium signaling and subsequent reduction in infarct size.

To record intracellular calcium in pyramidal neurons, researchers performed stereotaxic viral injections of GCaMP6m (AAV9-CaMKII-GCaMP6m) and then implanted an optical fiber above the injection site (Figure 1A, top). The initial recording location was chosen based on calibration experiments in the distal middle cerebral artery occlusion model (dMCAO), which showed that this area of the cortex was located in the infarct center. After 2-3 weeks of recovery, the second phase (Figure 1A, middle) included the recording day. Briefly, after baseline photometric recordings, mice were injected with vehicle or MK-801. After 15 minutes, mice were taken to the dMCAO operating room, and recordings continued for the next 3 hours. After 24 hours, in the third phase (Figure 1A, bottom), TTC staining was performed on serial brain sections. The infarct was fully formed in the dMCAO model at the 24-hour time point.

At 24 h following artery occlusion, the volume of the infarct tissue extended to 7 mm from rostral to caudal and covered most of the somatosensory cortex (Figure 1B). Interestingly, the researchers found that the overall infarct size was significantly larger in CRLC57 mice compared to the JaxC57 substrain (Figure 1C). Investigating the infarcted tissue section by section, the data showed that although the infarct size was significantly larger in CRLC57 mice from rostral to caudal, no interaction between mouse substrain and section position was found by two-way ANOVA (Figure 1D). These results indicate that despite the overall differences in the size of the infarct tissue between the different mouse substrains, the infarct core (located in both groups in section 3) and the pattern of tissue damage were similar between the groups.

Figure 1. Experimental paradigm for fiber photometry recordings in freely moving mice immediately following permanent distal middle cerebral artery occlusion. (Nelson A N, et al., 2020)