In vivo imaging of the early embryonic cortex in rodents
Year: 2017
Type: dissertation
Abstract: Embryonic brain development is a highly dynamic period in human life. Any disturbances at this stage can cause life-long negative consequences, such as developmentally related diseases, including autism, schizophrenia, and bipolar spectrum disorders. During development the mother-embryo interface plays a crucial role in supplementing the growing organism with oxygen and nutrients, regulating chemical cues, and protecting it from infections and potentially hazardous compounds. However, most of the studies of embryonic brain development have utilized ex vivo systems such as slices and neuronal cultures. Despite the great value of the data obtained from ex vivo studies, they usually completely ignore the significance of the mother-embryo interaction. Thus, there is desperate need for novel preclinical models of embryonic development. In this thesis, we developed a novel technique for in vivo two-photon imaging in mouse embryos connected to the mother via umbilical cord. We developed the special chamber with polymer membrane allowing to keep embryos separately in the heated physiological solution while the umbilical connection to the anesthetized mother is preserved. We developed the protocol for stimulation of calcium activity using high-power laser radiation and studied the propagation of the resulting calcium waves in the mouse embryonic cortex in vivo under ketamine/xylazine anesthesia. We confirm the enhancing effect of caffeine on the evoked activity and the suppressing effect of the adenosine triphosphate (ATP) -receptor blockade, known from previous ex vivo studies. We analyzed the patterns of wave propagation and show the non-uniform spreading, which suggests the presence of differing connectivity patterns in the cortex already during the early stage of development. Further, we studied spontaneous calcium activity and cellular motility in the mouse embryonic cortex in vivo under light isoflurane anesthesia. We demonstrate two various patterns of ongoing activity: sporadic activation of single cells and correlated activity in the form of calcium waves. We show that blockade of N-methyl-D-aspartate (NMDA) receptors with ketamine inhibits the calcium activity in vivo, corresponding with the arrest of cellular motility. In the last part of the thesis, we studied the dynamics of the externally introduced substance to the mouse embryonic brains in vivo. We used porous silicon nanoparticles, which are a promising drug delivery platform, as they can be loaded with poorly water-soluble drugs. We show that the nanoparticles can breach the placental barrier and accumulate in the brains of the embryo. To study the dynamics of nanoparticles when already in the cortex, we injected the embryonic brains intraventricularly. Nanoparticles, including ones 3-4 um in size, were distributed in 80% of the cortex already 4 hours following the injection, thus demonstrating high motility in the brain tissue of embryos. We confirmed the motility of nanoparticles in real time using the in vivo two-photon imaging of embryos connected to the mother under ketamine/xylazine anesthesia. The results emphasize the susceptibility of the embryonic cortex at the early stage of development to external particles, which should be taken into account in nanomedicine development. In summary, the developed in vivo imaging technique allowed functional studies in the embryonic cortex in real time. This will allow preclinical pharmacological investigations of the compounds while maintaining the physiological mother-embryo interface.
Source: Työväentutkimus Vuosikirja
Author: Mikhail Yuryev
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2614994724