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Idée - Biomedical Imaging - # In vivo Spinal Cord Imaging Using Three-Photon Microscopy

Three-Photon Excited Fluorescence Microscopy Enables Comprehensive Imaging of Spinal Cord Vasculature, Neural Structure, and Inflammatory Response in Living Mice


Concepts de base
Three-photon excited fluorescence microscopy enables high-contrast, high-resolution imaging of blood flow, neural structure, and inflammatory response in the mouse spinal cord up to 550 μm in depth, providing a powerful tool to study diverse cellular dynamics in the spinal cord in vivo.
Résumé

The study demonstrates that three-photon excited fluorescence (3PEF) microscopy using a 1,320-nm excitation wavelength enables deep, high-contrast imaging of the mouse spinal cord, reaching depths of up to ~550 μm. This is a significant improvement over the ~150 μm depth limit of the more commonly used two-photon excited fluorescence (2PEF) microscopy.

Using 3PEF, the researchers were able to:

  1. Map the detailed vascular architecture of the spinal cord, from surface venules to deep arterioles, and quantify blood flow speeds across this microvascular network.

  2. Follow the rapid degeneration of neural processes and the inflammatory response of resident microglia after photothrombotic occlusion of a surface venule. They observed depth-dependent structural changes in neurites and dynamic interactions of perivascular microglia with the occluded vessel and its upstream branches.

  3. Visualize the cessation of blood flow, axonal dieback, myelin degeneration, and microglia invasion of the vessel lumen following the surface venule occlusion.

The greater imaging depth enabled by 3PEF opens up new possibilities for cell-resolved studies of diverse physiological processes and pathological responses within the spinal cord in vivo. This technique provides a powerful tool to investigate the functional dynamics and cell-cell interactions in the spinal cord under both normal and disease conditions.

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Stats
The average red blood cell flow speed was around 1 mm/s in capillaries, with modest increases in flow speed with increasing vessel diameter in arterioles, but little variation across different diameter venules. Neurite morphology changed from intact to swollen, broken, and disappeared over 2 hours after the surface venule occlusion, with a more pronounced trend toward decreasing intact neurites at greater depths. A proportion of perivascular microglia migrated toward, extended processes to, and even invaded the vessel lumen of capillaries upstream from the occlusion, which was associated with increased blood-spinal cord barrier leakage.
Citations
"3PEF imaging with AO may provide improved SBR and depth penetration beyond that shown here." "The greater imaging depth afforded by 3PEF imaging opens the door to cell-resolved studies of a spectrum of physiological processes in the spinal cord."

Questions plus approfondies

How could the use of adaptive optics further improve the depth and resolution of 3PEF imaging in the spinal cord?

Adaptive optics (AO) can significantly enhance the depth and resolution of 3PEF imaging in the spinal cord by correcting tissue-induced aberrations. By using AO, it is possible to compensate for optical distortions caused by the highly scattering nature of the white matter in the dorsal spinal cord. AO systems can dynamically adjust the wavefront of the excitation light to counteract these aberrations, leading to improved image quality, increased signal-to-noise ratio, and enhanced spatial resolution. With AO, it is possible to achieve diffraction-limited imaging performance, even at greater imaging depths. By correcting for optical aberrations, AO can optimize the focus of the excitation light, allowing for sharper and clearer imaging of cellular structures deep within the spinal cord. This can enable researchers to visualize fine neural processes, individual cells, and subcellular structures with unprecedented detail and clarity. Furthermore, AO can help maintain high image quality throughout the imaging depth, reducing the loss of contrast and resolution that typically occurs with increasing tissue depth. By dynamically adjusting the optical path to compensate for variations in tissue properties, AO ensures that the excitation light is focused precisely on the target structures of interest, leading to more accurate and reliable imaging results. In summary, the integration of adaptive optics into 3PEF imaging systems can significantly improve the depth and resolution of imaging in the spinal cord by correcting tissue-induced aberrations, optimizing the focus of the excitation light, and enhancing overall image quality.

How could the use of adaptive optics further improve the depth and resolution of 3PEF imaging in the spinal cord?

The depth-dependent differences in neural degeneration and microglia responses observed after the vascular occlusion in the spinal cord could be driven by several potential mechanisms: Metabolic and Oxygen Supply Variations: Deeper regions of the spinal cord may have different metabolic demands and oxygen supply compared to superficial regions. The occlusion of a surface venule could lead to more severe ischemia at greater tissue depths, resulting in faster neural degeneration and more pronounced microglia responses. Cellular Vulnerability: Fine neural processes in the grey matter at greater depths may be more susceptible to ischemic conditions compared to larger axons on the dorsal surface. This differential vulnerability could contribute to the depth-dependent differences in neural degeneration observed after the vascular occlusion. Microenvironment Differences: The microenvironment in deeper laminae of the spinal cord may have distinct characteristics that influence cellular responses to injury. Factors such as local inflammation, immune cell distribution, and blood flow dynamics could vary with tissue depth, impacting the extent and nature of neural degeneration and microglia reactions. Axonal Architecture: The organization and connectivity of neural circuits within different laminae of the spinal cord may influence the propagation of degenerative processes and the recruitment of microglia. Variations in axonal architecture and synaptic connectivity could contribute to the observed depth-dependent differences in cellular responses. Blood-Spinal Cord Barrier Integrity: Differences in blood-spinal cord barrier integrity at varying tissue depths could affect the infiltration of immune cells, the leakage of blood components, and the overall tissue response to vascular occlusion. Changes in barrier function may modulate the extent of neural damage and microglia activation in a depth-dependent manner. By considering these potential mechanisms, researchers can gain insights into the complex interplay of cellular and molecular processes underlying the depth-dependent differences in neural degeneration and microglia responses observed in the spinal cord after a vascular occlusion.

What other cellular and functional dynamics in the spinal cord could be investigated using this 3PEF imaging approach, and how might the insights gained lead to a better understanding of spinal cord physiology and pathology?

The 3PEF imaging approach opens up a wide range of possibilities for investigating various cellular and functional dynamics in the spinal cord, including: Neuronal Activity: Real-time imaging of neural activity in different spinal cord regions can provide insights into sensory processing, motor control, and network dynamics. Monitoring calcium signaling, synaptic activity, and neuronal firing patterns using 3PEF can help unravel the mechanisms underlying spinal cord function. Immune Cell Interactions: Studying the interactions between microglia, astrocytes, and infiltrating immune cells in response to injury, infection, or neuroinflammation can shed light on the role of immune responses in spinal cord pathology. Visualizing immune cell dynamics and inflammatory processes can aid in understanding neuroinflammatory conditions. Vascular Dynamics: Investigating blood flow patterns, vascular permeability, and microcirculation in the spinal cord can provide valuable information about vascular health, oxygen delivery, and nutrient supply to neural tissues. Monitoring changes in blood flow speed, vessel morphology, and capillary integrity can help identify vascular contributions to spinal cord disorders. Axonal Regeneration: Tracking axonal sprouting, regeneration, and synaptic remodeling after injury or disease can offer insights into the mechanisms of neural repair and plasticity in the spinal cord. Visualizing axonal growth cones, collateral branching, and synapse formation using 3PEF imaging can inform strategies for promoting recovery. Glial Responses: Examining the activation, migration, and phagocytic activity of glial cells such as microglia, oligodendrocytes, and astrocytes in pathological conditions can elucidate their roles in neuroprotection, neuroinflammation, and tissue repair. Monitoring glial dynamics and interactions with neurons can provide clues to spinal cord homeostasis and dysfunction. By investigating these cellular and functional dynamics in the spinal cord using 3PEF imaging, researchers can advance our understanding of spinal cord physiology and pathology. The insights gained from such studies can inform the development of novel therapeutic strategies, diagnostic tools, and interventions for spinal cord injuries, neurodegenerative diseases, and other spinal cord disorders.
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