Chronic in vivo imaging in the mouse spinal cord using an implanted chamber

Abstract

Understanding and treatment of spinal cord pathology is limited in part by a lack of longitudinal in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. Repeated imaging was routinely achieved for more than five weeks post-operatively with up to ten separate imaging sessions. We observed neither motor function deficit nor neuropathology in the spinal cord as a result of chamber implantation. Using this chamber we quantified microglia and afferent axon dynamics following a laser-induced spinal cord lesion and observed massive microglia infiltration within one day along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

A Procedure for Implanting a Spinal Chamber for Longitudinal <em>In Vivo </em>Imaging of the Mouse Spinal Cord

“…Microglia were also visualized in mice expressing GFP in a CX3CR1 knock-in mouse (CX3CR1-GFP; Jackson Labs) crossed to the YFPH line ( Figure 4C). Contrast and resolution deteriorated modestly over time ( Figure 4C) due to the formation of a fibrous overgrowth described previously 30 . We routinely imaged for 3 hr in a single session without mortality and as often as every 12 hr.…”

“…For windows that remain clear, most places within the viewing window experience only a modest degree of neovascularization in or above the dura (Figure 3D -F, green asterisks), which does not hinder 2PEF imaging. Our previous study 30 showed an increase in microglia but not astrocyte density under the window and adjacent vertebrae, indicating mild inflammation analogous to that seen in cranial window preparations 5 .…”

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo

“…We have addressed the veracity of axonal fragmentation apparent from in vivo fluorescent microscopic observations in our previous papers 16,34,40 . The main arguments for our interpretation that clearly identifiable gaps in fluorescence contiguity correspond to actual axon discontinuities are: (1) in chronic experiments, where such gaps were identified by us 16 and others 41 , subsequent Wallerian degeneration of the axon segment distal to the gap in fluorescence was observed. (2) If combined with functional sensors, injuries that cause gaps in fluorescence generally correspond with signals expected to arise from plasma membrane disruptions (for example, ion influx, mitochondrial damage and so on) that spread in a predictable pattern away from the gap 42 .…”

Neutrophils Mediate Blood–Spinal Cord Barrier Disruption in Demyelinating Neuroinflammatory Diseases

“…This enables collecting data about cellular infiltration and status of the BSCB in asymptomatic animals and relating them to the exact timing of disease onset. Chronic implants have been described and would be the optimal strategy to put forward to remedy the situation, because they allow repetitive imaging of the same animal in a longitudinal fashion (52,53). However, in our hands they are not suitable in the study of autoimmune diseases because they require animals to be administered with immunosuppressant or anti-inflammatory drugs to limit fibrosis over the surface of the spinal cord, a critical step to achieve acceptable success rates and in-depth high-resolution imaging.…”