Long‐term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows

Abstract

Key points• Chronic in vivo imaging of cellular interactions within the adult spinal cord with subcellular resolution is important for understanding cellular physiology and disease progression.• Previous approaches for chronic in vivo spinal cord microscopy have required surgery for each imaging session.• Here we describe a novel method for implanting glass windows over the exposed spinal cords of adult mice for repeated in vivo microscopy.• We show that the windows remain clear for many months after implantation, do not damage axons or blood vessels, and are useful for studying cellular dynamics after spinal cord injury.• Our method represents an original technical breakthrough for scientists involved in spinal cord research and in vivo imaging, and is a useful tool for studying cellular physiology and disease progression.Abstract Repeated in vivo two-photon imaging of adult mammalian spinal cords, with subcellular resolution, would be crucial for understanding cellular mechanisms under normal and pathological conditions. Current methods are limited because they require surgery for each imaging session. Here we report a simple glass window methodology avoiding repeated surgical procedures and subsequent inflammation. We applied this strategy to follow axon integrity and the inflammatory response over months by multicolour imaging of adult transgenic mice. We found that glass windows have no significant effect on axon number or structure, cause a transient inflammatory response, and dramatically increase the throughput of in vivo spinal imaging. Moreover, we used this technique to track retraction/degeneration and regeneration of cut axons after a 'pin-prick' spinal cord injury with high temporal fidelity. We showed that regenerating axons can cross an injury site within 4 days and that their terminals undergo dramatic morphological changes for weeks after injury. Overall the technique can potentially be adapted to evaluate cellular functions and therapeutic strategies in the normal and diseased spinal cord.

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.…”

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

“…Over time, this growth resulted in the loss of image contrast and resolution. This growth has also been seen in alternative surgical preparations 31 . Anecdotally, we have observed that this growth can be minimized by carefully washing the dorsal SC surface to remove blood products, sealing the surface of the cut bone with cyanoacrylate, sealing edges of the chamber well with silicone elastomer, and minimizing the interstitial space between the dorsal SC and the cover glass.…”

Tests of isotropy for rough textures of trended images

“…Hence, results obtained for these cases could have been better if we had chosen to simulate fields with higher amplitudes. (Fenrich et al (2012(Fenrich et al ( , 2013). …”