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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Laminectomy and Spinal Cord Window Implantation in the Mouse

Published: October 23rd, 2019

DOI:

10.3791/58330

1Department of Anatomy and Cell Biology, University of Illinois at Chicago College of Medicine, 2Medical Scientist Training Program, University of Illinois at Chicago College of Medicine

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

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.

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....

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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.......

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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.......

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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.......

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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....

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Name Company Catalog Number Comments
3D printer Raise3D Pro2 For printing backplates
PLA 3D printing filament Inland PLA+-175-B Black plastic 3D printing material
3D CAD software Dassault Systemes Solidworks software used to design 3D shapes
3D printer software Raise3D Ideamaker software software used to interface with the 3D printer
3D printed oval backplate custom Stabilizing imaging field
Surgical dissecting microscope Leica M205 C Equipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage
Microscope camera Leica MC170 HD color camera for visualizing surgical field
Gliding stage Leica 10446301 The 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 fork Whale Manufactoring custom Laminectomy
SomnoSuite low-flow isoflurane delivery unit Kent Scientific SS-01 Surgical anesthesia administration with integrated digitial vaporizer
Stainless steel 1.5 inch mounting post ThorLabs P50/M For mounting surgical station onto optical table for two-photon imaging
Counterbored Clamping Fork for 1.5" mounting Post ThorLabs PF175 For stabilizing surgical station mount onto optical table for two-photon imaging
Ideal bone microdrill Harvard apparatus 72-6065 Thinning bone for laminectomy
Water bath Fisher Scientific 15-462-10 Warming saline
Cautery gun FST 18010-00 Cauterizing minor bleeds
Heating pad Benchmark BF11222 1.9” x 4.5” silicone heater with 20” Teflon leads, 10W, 5V
K type thermocoupled rectal probe Physitemp RET3 Measuring mouse body temperature
petroleum jelly Sigma 8009-03-8 Lubricating rectal probe
Feedback-regulated thermal controller custom NA Commercially available alternatives include the Physitemp TCAT series
PVA Surgical eye spears Beaver-visitec international 40400-8 Absorbing blood
Electric trimmer Wahl 41590-0438 Trimming mouse fur
Blade, #11 FST 14002-14 Surgical tool
Forceps, #5 FST 11254-20 Surgical tool
Forceps, #4 FST 14002-14 Surgical tool
Titatnium toothed forceps WPI 555047FT Surgical tool
Titanium Iris scissors WPI 555562S Surgical tool
Vetbond tissue adhesive 3M 084-1469SB Preparing tissue surface for dental acrylic
Ceramic mixing tray Jack Richeson 420716 Mixing dental acrylic agent with accelerant
Orthojet dental acrylic Lang Dental 1520BLK, 1503BLK Permanently bonding backplate to tissue
Small round cover glass, #1 thickness, 3 mm Harvard apparatus 64-0720 optical window
NaCl Fisher Scientific 7647-14-5 For aCSF
KCl Fisher Scientific 7447-40-7 For aCSF
Glucose Fisher Scientific 50-99-7 For aCSF
HEPES Sigma 7365-45-9 For aCSF
MgCl2·6H2O Fisher Scientific 7791-18-6 For aCSF
CaCl2·2H2O Fisher Scientific 10035-04-8 For aCSF
Carprofen Rimadyl QM01AE91 Analgesia
Bacteriostatic water Henry Schein 2587428 Diluent for carprofen
Isoflurane Henry Schein 11695-6776-2 Anesthesia
Lactated ringer solution Baxter 0338-0117-04 Hydration for mouse
Agarose High EEO Sigma A9793 gel point 34-37 degrees C
Opthalmic lubricating ointment Akwa Tears 68788-0697 Prevent corneal drying
MOM Two-Photon Microscope Sutter

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