Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows - PubMed (original) (raw)

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

Keith K Fenrich et al. J Physiol. 2012.

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.

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Figures

Figure 1

Figure 1. Spinal glass window implantation procedure

A, schematic diagram showing the vertebral clamp and laminectomy. The tips of the vertebral clamp (inset; magenta arrowheads) were inserted under the vertebral transverse processes and glued into place with cyanoacrylate. B, image showing a modified paperclip. The lower parts, including the tips, were implanted into the dental cement surrounding the window. The upper part protruded from dental cement and was used to clamp the animal for surgery and imaging. Scale bar, 1 cm. C, image of an anaesthetized mouse with an implanted window. The window structure is being supported by a clamp attached to a plastic base. The animal is freely breathing while in the support, but the window remains stationary relative to the clamp. D, detailed view of spinal glass window (from boxed region in A). The edges of the vertebrae were shaped so that the window rests on both sides of the vertebral opening without compressing the spinal cord. Kwik-Sil was used as a physical barrier preventing the infiltration of opaque tissue between the window and spinal cord.

Figure 2

Figure 2. Spinal glass window viability and basic properties

A, plots showing animal weight over time as a percentage change from before surgery. Average (black lines) and data from individual animals (grey lines) (n = 17). Error bars, SEM. B and C, representative images showing the same spinal cord region immediately before (B), and after (C) window implantation. The blue arch in the top of B (SHG, second harmonic generation from collagen in dura mater) highlights the curvature of the spinal cord before window implantation. Arrows in B show the horizontal distortions in the image caused by respiratory movements before window implantation. Scale bar, 100 μm. Excitation wavelength 945 nm. D_–_F, images of a spinal cord through a spinal glass window 28 days after implantation. D, image showing exposed spinal cord through window. Dashed box outlines the area shown in E. Scale bar, 1 mm. E, representative images of axons (Thy1), LysM(+) cells, and blood vessels (Rhodamine). Dashed box outlines the area shown in F. Scale bar, 50 μm. F, higher magnification image showing detailed structure of individual axons, blood vessels, a LysM(+) cell within the blood vessel (arrow head), and perivascular LysM(+) cells (arrows). Notice that the perivascular cells also contain Rhodamine, likely to have been engulfed following previous imaging sessions. Scale bar, 20 μm. G and H, images showing axons and blood vessels from the same spinal region at 0 days and 350 days after window implantation (n = 22 imaging sessions). The same axon (arrowheads) was identified at each subsequent post-implantation interval. Scale bar, 50 μm. I, high power view of identified axon at 350 days post-implantation. Scale bar, 50 μm. Excitation wavelength 945 nm. In all panels Caudal is up, Rostral is down.

Figure 3

Figure 3. Long-term spinal cord integrity after glass window implantation

A, representative images of the same region of a Thy1-CFP//LysM-GFP mouse spinal cord (4–28 days) after window implantation. Upper left dashed boxes indicate the regions used for axon counts for all subsequent imaging sessions (see G). The white lines across the blood vessels outline the site of blood vessel diameter measurements (see H). The same blood vessels were measured at 5 locations for every session. Bottom left dashed box shows the region used to count LysM(+) cells (see E and F). Excitation wavelength, 840 nm (top row) and 985 nm (bottom row). Scale bar, 100 μm. B, representative images showing the same regions of a Thy1-CFP//CD11c-YFP mouse spinal cord (0–30 days) after window implantation. Excitation wavelength, 945 nm. Scale bar, 50 μm. C and D, representative images showing the morphological characteristics of LysM(+) (C) and CD11c(+) (D) cells at 18d and 26d respectively. Excitation wavelength, 985 nm (C) 945 nm (D). Scale bars, 20 μm. E and F, average number (black line) of LysM(+) (E) and CD11c(+) (F) cells within a predefined zone of the spinal cord (dashed box in lower left image of A) over time. Grey lines, data from individual animals. Error bars, SEM (n = 3 LysM-GFP; n = 3 CD11c-YFP). G, mean number of axons (black line) within predefined zones of the spinal cord (dashed boxes in upper left image of A) over time. Grey lines, data from individual animals (n = 6). Error bars, SEM. H, plots showing the average diameter of central vein side branches (black line) over time. Grey lines, data from individual animals (n = 6). Error bars, SEM. I and J, representative images showing the same regions of a Thy1-GFP mouse after multiple imaging sessions using QDot-655 to visualize the vascular compartment. Notice the lack of QDot-655 labelled perivascular cells after 5 (I) and 8 (J) imaging sessions (cf. A_–_D). Excitation wavelength, 840 nm. Scale bars, 50 μm. In all panels Caudal is up, Rostral is down.

Figure 4

Figure 4. Axon dynamics after spinal cord injury

A, representative image showing axons and blood vessels 12 days after injury in a Thy1-CFP mouse. Scale bar, 100 μm. B_–_F, quantification of axon loss and recruitment at injury sites (B), two zones caudal (C and D), and two zones rostral (E and F) of the injury sites. Averages across animals (black lines); data from individual animals (grey lines). Error bars, SEM (n = 7). G, image stacks showing an injury site (orange star) in a Thy1-CFP mouse over time. Green arrowheads at 4d, 6d, 8d, and 26d mark the trajectory of an axon that crossed the injury site between 0d and 4d (green and red shaded axons in lower panels of I respectively). Scale bar, 100 μm. H and I, image stacks showing a cut axon terminal elongating caudally over time (H; outlined by the magenta box in G), and a cut axon whose primary shaft crossed the injury site between 0d and 4d (green arrowheads in G; green shaded axon in I). Lower panels show coloured tracings of the axons shown in dashed boxes of the upper panels. Yellow arrows (I) indicate that the axon continued beyond the end of the tracing, but was too deep within the tissue or entangled within other CFP axons to be resolved. Scale bars, 40 μm. J, image stacks showing two uninjured axons (arrows) and a cut axon terminal (arrowheads) caudal of an injury site in a Thy1-GFP mouse over time. This terminal remains swollen, has no branches, does not regenerate, and progressively retracts over time. Scale bars, 50 μm. In all panels Caudal is up, Rostral is down.

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