In vivo imaging study of spinal cord injury based on advanced multimodal nonlinear optical microscopy

Abstract

Seeing is believing. In vivo optical imaging allows us to visualize the complex microenvironment in biological systems more completely and with less bias. Imaging live animals over time is an effective and straightforward way to study the real-time physiology and pathology involved in various disease progressions including spinal cord injury (SCI). In particular, nonlinear optical (NLO) microscopy has become a valuable tool in biological research in recent decades, thanks to its numerous unique advantages. Unlike conventional optical microscopy, NLO microscopy derives its contrast from multiple nonlinear optical processes, making it possible to perform multimodal imaging with exceptional specificity and selectivity. Each type of nonlinear optical signal provides insight into specific properties of biological tissue structures and functions. Additionally, NLO microscopy offers a large penetration depth as it uses longer-wavelength near-infrared light to excite nonlinear optical signals. Furthermore, it enables inherent three-dimensional (3D) imaging, providing better image contrast and decreased out-of-focus photodamage and photobleaching. Due to its unique properties, NLO microscopy has become the preferred choice for in vivo imaging studies. Given the numerous advantages of NLO microscopy techniques, my thesis work primarily focuses on using the multimodal NLO technologies to study cellular dynamics and interactions in healthy and injured spinal cord, which may offer clues to develop effective treatment for spinal cord injury. Specifically, we developed a minimally invasive spinal cord window that allows for visualizing cellular activities in the spinal cord without introducing inflammatory responses. Based on an inherent intervertebral window, we further retained the ligamentum flavum layer and introduced an optical clearing method to achieve minimally invasive and high-resolution imaging in a repetitive and longitudinal way. With the advanced intervertebral window, we next investigated the response of myelinated axons and microglia to laser induced injury in spinal dorsal column. While the dorsal column is mostly filled with longitudinally running myelinated axons, the dorsal horn region contains neuronal cell bodies as well as dendrites, functioning as an intermediate processing center for somatosensory and viscerosensory information. Despite the essential function of dorsal horn, in vivo imaging at this region is quite challenging as it is buried under the highly scattered superficial axon tracts. Through the intervertebral window, we identified a thin-myelin region that allows imaging over 200 μm deep below pia, well into the spinal dorsal horn. By using optical clearing, we finally achieved repetitive dorsal horn imaging in a minimally invasive way over a period of time. To go one step further, with the well-established in vivo imaging method, we explored the microglia-axon interaction at the nodes of Ranvier in normal and injured spinal cord. We observed specialized wrapping contact at nodes by microglial processes in response to axonal injury, which depends on microglial P2Y12 receptor activity. Interestingly, this wrapping contact was proven to stop axonal acute degeneration at or slightly before the nodes of Ranvier, showing the neuroprotective role of microglia in injured spinal cord, which may be considered a new therapeutic target to develop effective treatment strategies.

Date of Award	2023
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Jianan QU (Supervisor)