Instructor Cincinnati Children's Hospital Med Ctr Cincinnati, Ohio
Rationale: Cortical spreading depolarizations (SDs) have been associated with a variety of brain pathologies, including seizures. SDs propagate through cortex as a wave, depolarizing neurons and temporarily shutting down the network. Their long-term consequences are largely unknown, partly due to the difficulty of studying them in vivo. Often, multiple electrodes are used to capture the cortical depression propagating through the tissue, providing a point by point view of electrical changes. Advances in imaging techniques, like calcium imaging (CI), allows the viewing of neuron populations during SDs, but many are only applicable in animal models. Optical intrinsic signal imaging (OISI) provides a means of viewing large areas of live cortex without contrast agents and is therefore amenable to clinical implementation. However, since OISI partially relies on hemodynamic responses, we hypothesized that timing and spatial representation of responses would vary from the neural activation detected with CI. To better understand the relationship between neuronal polarization states and OISI, we examined simultaneous OISI data and neuronal activation in transgenic GCaMP6 mice during SDs. Methods: Thy1-GCaMP6f (n=18) heterozygous C57Bl6 mice underwent surgery with isoflurane anesthesia to install a cranial window. Imaging took place on a Nikon SpectraX system with ~10 µs fast gating acquisition of 488 nm fluorescence and reflected light from 555 and 640 nm channels for OISI. A capillary tube pulled to a sharp tip was inserted into the cortex to induce a SD on a subset of mice (n=10). Isoflurane was increased to 5% to investigate ischemia induced terminal SDs in all mice. Results: Mechanical stimulation and isoflurane induced ischemia consistently initiated SDs. SDs traveled with consistent velocities for up to 500 µm from the initiation site, measured every 100µm. However, SD spread velocity was faster when analyzing GCaMP activation compared OISI (mean ± SEM mm/min: 470nm, 4.52±0.16; 555nm, 2.80±0.12; 640nm, 2.00±0.19; F2=70.6, p< 0.001). SD initiation was from the same location in the tissue, regardless of wavelength analyzed, but the SD was detected by CI significantly sooner compared to OISI (555nm: 7.63±1.45s later than 470nm; 640nm: 10.76 ±1.23s after 470nm; F2=45.28, p< 0.001, RM ANOVA). Conclusions: Fast gated three-channel imaging allowed the characterization of neuronal activation and local tissue oxygenation levels from the simultaneous OISI during SDs. While the CI signal propagation through the cortex and OISI data match previously reported SD velocity and alterations in blood oxygenation, the SD initiation and propagation seen with CI was earlier and faster than OISI changes. The spatial accuracy of OISI was consistent with CI. Other patterns in the reflected light channels are being investigated to determine if OISI could be utilized to anticipate SDs or seizures in epilepsy models. Funding: Please list any funding that was received in support of this abstract.: Support provided by Division of Neurosurgery, Cincinnati Children’s Hospital Medical Center.
