Published: October 23rd, 2019
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....
All experiments follow the University of Illinois, Chicago Institutional Animal Care and Use Committee protocols. This is a terminal procedure.
1. Reagent Preparation
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.......
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.......
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....
|For printing backplates
|PLA 3D printing filament
|Black plastic 3D printing material
|3D CAD software
|used to design 3D shapes
|3D printer software
|software used to interface with the 3D printer
|3D printed oval backplate
|Stabilizing imaging field
|Surgical dissecting microscope
|Equipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage
|HD color camera for visualizing surgical field
|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
|SomnoSuite low-flow isoflurane delivery unit
|Surgical anesthesia administration with integrated digitial vaporizer
|Stainless steel 1.5 inch mounting post
|For mounting surgical station onto optical table for two-photon imaging
|Counterbored Clamping Fork for 1.5" mounting Post
|For stabilizing surgical station mount onto optical table for two-photon imaging
|Ideal bone microdrill
|Thinning bone for laminectomy
|Cauterizing minor bleeds
|1.9” x 4.5” silicone heater with 20” Teflon leads, 10W, 5V
|K type thermocoupled rectal probe
|Measuring mouse body temperature
|Lubricating rectal probe
|Feedback-regulated thermal controller
|Commercially available alternatives include the Physitemp TCAT series
|PVA Surgical eye spears
|Trimming mouse fur
|Titatnium toothed forceps
|Titanium Iris scissors
|Vetbond tissue adhesive
|Preparing tissue surface for dental acrylic
|Ceramic mixing tray
|Mixing dental acrylic agent with accelerant
|Orthojet dental acrylic
|Permanently bonding backplate to tissue
|Small round cover glass, #1 thickness, 3 mm
|Diluent for carprofen
|Lactated ringer solution
|Hydration for mouse
|Agarose High EEO
|gel point 34-37 degrees C
|Opthalmic lubricating ointment
|Prevent corneal drying
|MOM Two-Photon Microscope
Copyright © 2024 MyJoVE Corporation. All rights reserved