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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Abstract

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 — a tight junction protein.

Introduction

Transgenic animal models expressing fluorescent proteins, when combined with intravital microscopy, provide a powerful platform for addressing biology and pathophysiology. To apply these techniques to the spinal cord, specialized protocols are required to prepare the spinal cord for imaging. One such strategy is to conduct a laminectomy and spinal cord window implantation. The key features of an ideal laminectomy protocol for microscopy include preservation of native tissue structure and function, stability of the imaging field, quick processing time, and reproducibility of results. A particular challenge is to stabilize the imaging field against the motion induced by....

Protocol

All experiments follow the University of Illinois, Chicago Institutional Animal Care and Use Committee protocols. This is a terminal procedure. 

1. Reagent Preparation

  1. Prepare artificial cerebral spinal fluid (aCSF) to contain 125 mM NaCl, 5 mM KCl, 10 mM Glucose, 10 mM HEPES, 2 mM MgCl2·6H2O, 2 mM CaCl2·2H2O in ddH2O. Sterile filter and freeze in individual-use aliquots. Warm aCSF in a water bath to 39 °C b.......

Representative Results

Implanted glass windows and intravital two-photon microscopy provides a useful tool for assessing dynamic changes in CNS proteins. The functional integrity of the BBB is influenced by the expression, subcellular localization, and turnover rates of tight junction proteins7. Previous studies have demonstrated that tight junction proteins undergo rapid and dynamic remodeling at steady state8. The currently described laminectomy and glass window.......

Discussion

The method described here allows for stable imaging of the spinal cord in mice through a glass window. This method has been applied to assess BBB remodeling in transgenic eGFP:Claudin5+/- mice that express a fluorescent BBB tight junction protein, but it could be applied equally well for studies of any fluorescent proteins or cells in the spinal cord.

Multiple methods for laminectomy and spinal cord stabilization have been developed. All protocols address stabilizing the spinal cord during ima.......

Acknowledgements

S.E. Lutz is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, under Grant KL2TR002002 and University of Illinois Chicago College of Medicine start-up funds. Simon Alford is supported by RO1 MH084874. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors thank Dritan Agalliu in the Department of Neurology at Columbia University Medical Center for the Tg eGFP:Claudin-5 mice, scientific discussions, and insights into the development of the surgical protocol and imaging applications. The authors thank Sunil P. Gandhi in the Department of Neu....

Materials

NameCompanyCatalog NumberComments
3D printerRaise3DPro2For printing backplates
PLA 3D printing filamentInlandPLA+-175-BBlack plastic 3D printing material
3D CAD softwareDassault SystemesSolidworks softwareused to design 3D shapes
3D printer softwareRaise3DIdeamaker softwaresoftware used to interface with the 3D printer
3D printed oval backplatecustomStabilizing imaging field
Surgical dissecting microscopeLeicaM205 CEquipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage
Microscope cameraLeicaMC170HD color camera for visualizing surgical field
Gliding stageLeica10446301The gliding stage is constructed of two metal plates. The base plate is fixed. The upper plate slides on greased interface to allow rotational and linear movement.
Surgical station and stabilization forkWhale ManufactoringcustomLaminectomy
SomnoSuite low-flow isoflurane delivery unitKent ScientificSS-01Surgical anesthesia administration with integrated digitial vaporizer
Stainless steel 1.5 inch mounting postThorLabsP50/MFor mounting surgical station onto optical table for two-photon imaging
Counterbored Clamping Fork for 1.5" mounting PostThorLabsPF175For stabilizing surgical station mount onto optical table for two-photon imaging
Ideal bone microdrillHarvard apparatus72-6065Thinning bone for laminectomy
Water bathFisher Scientific15-462-10Warming saline
Cautery gunFST18010-00Cauterizing minor bleeds
Heating padBenchmarkBF112221.9” x 4.5” silicone heater with 20” Teflon leads, 10W, 5V
K type thermocoupled rectal probePhysitempRET3Measuring mouse body temperature
petroleum jellySigma8009-03-8Lubricating rectal probe
Feedback-regulated thermal controllercustomNACommercially available alternatives include the Physitemp TCAT series
PVA Surgical eye spearsBeaver-visitec international40400-8Absorbing blood
Electric trimmerWahl41590-0438Trimming mouse fur
Blade, #11FST14002-14Surgical tool
Forceps, #5FST11254-20Surgical tool
Forceps, #4FST14002-14Surgical tool
Titatnium toothed forcepsWPI555047FTSurgical tool
Titanium Iris scissorsWPI555562SSurgical tool
Vetbond tissue adhesive3M084-1469SBPreparing tissue surface for dental acrylic
Ceramic mixing trayJack Richeson420716Mixing dental acrylic agent with accelerant
Orthojet dental acrylicLang Dental1520BLK, 1503BLKPermanently bonding backplate to tissue
Small round cover glass, #1 thickness, 3 mmHarvard apparatus64-0720optical window
NaClFisher Scientific7647-14-5For aCSF
KClFisher Scientific7447-40-7For aCSF
GlucoseFisher Scientific50-99-7For aCSF
HEPESSigma7365-45-9For aCSF
MgCl2·6H2OFisher Scientific7791-18-6For aCSF
CaCl2·2H2OFisher Scientific10035-04-8For aCSF
CarprofenRimadylQM01AE91Analgesia
Bacteriostatic waterHenry Schein2587428Diluent for carprofen
IsofluraneHenry Schein11695-6776-2Anesthesia
Lactated ringer solutionBaxter0338-0117-04Hydration for mouse
Agarose High EEOSigmaA9793gel point 34-37 degrees C
Opthalmic lubricating ointmentAkwa Tears68788-0697Prevent corneal drying
MOM Two-Photon MicroscopeSutter

References

  1. Weinger, J. G., et al. Two-photon imaging of cellular dynamics in the mouse spinal cord. Journal of Visualized Experiments. (96), (2015).
  2. Davalos, D., Akassoglou, K. In vivo imaging of the mouse spinal cord using two-photon m....

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