Neural tube closure (NTC) is a highly synchronized morphological process driven by mechanical forces, and any disruptions during this process can lead to neural tube defects (NTDs). However, mechanical properties associated with NTDs are largely unknown. To understand the correlation between NTDs and biomechanical properties, we imaged NTC using multimodal Brillouin microscopy and optical coherence tomography in two mutant mice lines, where the genes Mthfd1l and Fuz were inactivated. We also imaged cerebral organoids cultured in dolutegravir for 10 and 14 days. Our results showed a clear link between NTDs and neural tube biomechanical properties.
Neural tube closure is a complex process driven by mechanical forces, but this process can be disturbed leading to development defects. So, to understand the interplay between forces and tissue stiffness during neurulation, we developed a multimodal Brillouin microscopy and optical coherence system (OCT). OCT provides structural guidance while mapping the biomechanical properties of embryonic neural tube using Brillouin microscopy. 3D-OCT, 2D-OCT, and 2D-Brillouin images of Mthfd1l and Fuz knockout mouse embryos at gestation days 9.5 and 10.5 were acquired. Our results show overall decrease in the stiffness of homozygotic knockout neural tube tissues compared to the wildtype.
The mechanisms involved in neural tube formation are complex and can be easily disrupted. Neurulation is one such process, governed by mechanical forces where tissues physically fold and fuse. When neural tube folding and closure fail to complete during neurulation, it results in structural and functional abnormalities of the brain and spinal cord. Thus, it is important to understand the interplay between forces and tissue stiffness during neurulation. Brillouin microscopy is an all-optical, noninvasive, high-resolution imaging technique capable of mapping tissue stiffness, but it cannot provide structural information, resulting in “blind” imaging. To overcome this limitation, we have combined a Brillouin microscopy system with optical coherence tomography (OCT) in one synchronized and co-aligned instrument to provide structural guidance when mapping the biomechanical properties of neural tube formation in mouse embryos. We developed custom instrumentation control software that utilizes the OCT structural image to guide Brillouin imaging. We acquired first 3D OCT images and then 2D structural and mechanical maps of mouse embryos at embryonic day (E) 8.5, 9.5, and 10.5. Brillouin microscopy showed the cell-dense layer of neural plate derived from the ectoderm at E 8.5, which was unable to be distinguished with OCT. At E 9.5 and 10.5, the neuroepithelium could be clearly seen by Brillouin microscopy with a greater stiffness than the surrounding tissue. Our results show the capability of the co-aligned and synchronized Brillouin-OCT system to map tissue stiffness of murine embryos using OCT-guided Brillouin microscopy.
Embryonic development involves the interplay of driving forces that shape the tissue and the mechanical resistance that the tissue offers in response. While increasing evidence has suggested the crucial role of physical mechanisms underlying embryo development, tissue biomechanics is not well understood due to the lack of techniques that can quantify the stiffness of tissue in-situ with 3D high-resolution and in a non-contact manner. In this work, we used two all-optical technique, optical coherence tomography (OCT) and Brillouin microscopy, to map the longitudinal modulus of the neural tube tissue of mouse embryo in-situ. We found the tissue stiffens significantly after the closure of the neural tube at cranial regions by comparing embryos at E 8.5 and E 9.5. In addition, we observed that the region of fusion following neural tube closure is softer than the adjacent neural folds, and the neural folds show a modulus gradient along dorsal-ventral direction. Furthermore, we found the overlaying ectoderm is much softer and more pliable than the closed neural tube, and thus can be distinguished based on its mechanical properties. In conclusion, we demonstrated the capability of OCT and Brillouin microscopy to quantify tissue modulus of mouse embryos in-situ, and observed a distinct change of tissue modulus during the closure of cranial neural tube, suggesting this method could be helpful in investigating the role of tissue biomechanics in the regulation of embryo development.
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