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

Researchers Use Window on Live Mouse to View Spinal Cord Trauma http://www.news.cornell.edu/stories/Jan12/SpinalImaging.html
January 26, 2012 by Staff
Science
Cornell researchers are using exploratory surgeries on mice to further their understanding of spinal cord injuries. By surgically implanting a window with a transparent panel over a live mouse’s exposed cord, the researchers are able to study how nerves and other cells respond after trauma.

A spinal chamber implantation enables long term optical imaging of the mouse spinal cord. A 3-piece spinal chamber (top) which attaches to the mouse vertebral column enables scientists to see into the spinal cord of mice without having to perform multiple painful and potentially complicating surgeries. Transgenic mice expressing fluorescent proteins combined with other labeling strategies allow for the direct and long-term visualization of cell types and structures.

To study spinal cord injuries, researchers have had to conduct exploratory surgeries on mice to determine how nerves and other cells respond after trauma. But these approaches have only shown snapshots in time and have failed to provide key, game-changing information.
But now, Cornell researchers have developed an imaging technique similar to one used to study the brain, in which a small portion of a mouse skull is replaced with glass so scientists can observe the brain to study Alzheimer’s disease, strokes and brain cancers.
The new spinal cord procedure, described in the Jan. 22 online issue of the journal Nature Methods, involves surgically implanting a window, or chamber with a transparent panel over a live mouse’s exposed cord. Fluorescently labeled objects, such as neural cells or blood vessels, are then visualized in 3-D using multiphoton microscopy (invented by Cornell biophysicist Watt W. Webb).
The implant has allowed researchers to image the spinal cord for more than five weeks without affecting motor function or causing nerve damage. Rods in the chamber allow researchers to suspend the mouse above the operating table, so breathing and other movements do not interfere with the imaging.

Using the new chamber, researchers imaged axons (green) and blood vessels (red) in a mouse’s spinal cord.

“With this procedure, we can study the same animal and look at whether axons (nerve cells) are dying back [gradual dying, beginning at the tips] or growing over time,” said Chris Schaffer, associate professor of biomedical engineering and the paper’s senior author. “It opens the door to new classes of experiments.”
“Our method provides a platform for rapid evaluation of the efficacy of different therapeutic strategies,” said Matthew Farrar, a graduate student in Schaffer’s lab and the study’s lead author. Joseph Fetcho, a professor of neurobiology and behavior, is also a co-author.
The new method allows continuous observations of axon behavior. Previously, researchers had to conduct repeated surgeries to check the status of axons, which provided only a snapshot of the healing process and made it difficult to determine if axons were growing or dying back.
Preliminary data confirm previous observations that the inflammatory cells of the spinal cord (microglia) surge in number immediately after trauma, while axons show varied behavior — some begin to die back while others soon start to regrow, but the growth tapers off after eight days. Such insights may help researchers determine when and where to focus efforts when it comes to regrowing severed nerve cells.
“Axons that manage to persist near the injury may have the best chance of regrowing,” said Schaffer. “Growing back every axon is probably not necessary for some recovery of function, and getting back just a few may be valuable.”
Also, the new procedure offers insights into the optimal time for treating a spinal injury. The priority for clinicians has been to stabilize a patient immediately following spinal trauma, but they may want to also apply therapies early in the recovery process, Farrar added.
Without continuous long-term observations, early readings also may lead one to “falsely conclude that a therapy is more successful than it really is,” said Farrar.
Source: Krishna Ramanujan, Cornell University

Chronic in vivo imaging in the mouse spinal cord using an implanted chamber
Journal name:Nature MethodsYear published2012)DOI:doi:10.1038/nmeth.1856Received10 March 2011 Accepted08 December 2011 Published online22 January 2012 Abstract

    • Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse 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. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and 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 after a laser-induced spinal cord lesion and observed massive microglia infiltration within 1 d 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.

Figures at a glance

  • Figure 1: An imaging chamber for longitudinal optical access to mouse spinal cord without the need for repeated surgeries.  (a) Photograph of the imaging chamber. (b) Schema showing the implantation of the imaging chamber in mice at the T11–T12 vertebra, just below the dorsal fat pad (taupe). (c) Photograph showing the spinal cord imaged through the implanted chamber 144 d after the surgery. (d) Photograph of a mouse with an implanted chamber (same mouse as in c).
  • Figure 2: Longitudinal 2PEF imaging of axons and blood vessels over many weeks after surgery.  (a) Projections of 2PEF image stacks of afferent axons expressing EYFP (teal) and blood vessels labeled with intravenously injected Texas Red dextran (red) taken over 9 weeks after chamber implantation. Asterisks indicate the location of red autofluorescence from invading, likely inflammatory, cells located above the spinal cord at later time points. Arrows denote landmark features of the axons that were visible at all time points. (b) High-resolution 2PEF imaging of EYFP-expressing axons from the same region as in a. (c) Profile and fit (Online Methods, equation (3)) across maximum intensity projections of selected axon segments shown in the boxed region in b and in the inset; scale bar, 30 μm). A.u., arbitrary units. (d,e) Image contrast (d) and lateral spatial resolution (e) as functions of time after surgery from the fits for all axon segments for two mice (separate curves for each mouse, ~10 axons measured at each time point for each mouse). Error bars, s.d.
  • Figure 3: Histological analysis of reactive microglia and astrocytes, and tissue morphology after chamber implantation.  (a,b) Wide-field fluorescence images of 30-μm-thick coronal tissue sections from the laminectomy site 1 d and 1 week after implantation and in non-surgical controls for mice expressing EGFP in microglia (a) or astrocytes (b). (c) Hematoxylin and eosin–stained tissue section taken 7 d after implantation. Magnifications of the left and right boxed regions show the fibrous connective tissue that covered the dorsal aspect of the spinal cord under the implant and the neural tissue, respectively. (d,e) Microglia (d) and astrocyte (e) densites in spinal cord sections 1 and 7 d after implantation for sections one vertebra rostral to the surgical site (rost.), at the surgical site (surg.) and one vertebra caudal to the surgical site (caud.) and in controls (*P = 0.012; **P = 0.0010; ***P = 0.0098; #P < 0.0001; n ≥ 15 measurements per segment per time point; 3 mice per time point). Error bars, s.e.m.
  • Figure 4: Imaging and quantification of microglial scar formation at the site of a laser-induced SCI.

http://scitechdaily.com/researchers-…l-cord-trauma/http://www.nature.com/nmeth/journal/…meth.1856.html

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