Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Summary

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Abstract

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.

Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

Introduction

Multiple sclerosis is a CNS auto-immune disease that causes progressive myelin and axon damage. The standard research model for CNS autoimmune demyelinating diseases is EAE in adult rodents. However, the dynamic cellular events underlying EAE progression remain largely unexplored. One approach is to use intravital two-photon microscopy to visualize cellular populations with fluorescent markers for extended periods of time in the spinal cords of adult mice with EAE.

Intravital two-photon microscopy is a powerful way to study cellular physiology, cellular interactions, and the dynamic progression of disease in living animals with subcellular ....

Protocol

1. Window and Support Structure Preparation

1. Window preparation - prepare several windows of various sizes (width = 1.8-2.2 mm; length = 4.5-5.2 mm) for implantation.
   1. Making the modified drill burr to cut the cover glass - using a generous amount of dental cement attach a drill burr to a shaft (in our case old Bulldog clamps). Be sure that the burr is ~45° relative to the shaft for easier handling.
      Note: diamond scribes also work well for cutting cover glass.
   2. Making slide-A - using a standard microscope slide, tape a short piece of ruler paper (~2 cm) to the bottom of the slide so that you can see the ruler through the gl....

Results

Using chronically implanted glass windows in combination with intravital two-photon microscopy and EAE models may be a useful tool to understand the cellular mechanisms underlying autoimmune demyelinating disease pathology. Using our implantation method we found that most windows were clear at the time of EAE induction, and remained clear throughout EAE progression (Figure 1c). Using this approach, we found that windows remained clear for the entire postimplantation period (average ~68 d±7 s.e.m.; n = 21.......

Discussion

We described a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice to study the dynamic progression of EAE over time. A similar glass window implantation approach has been used to study spinal cord injury models¹³ and may also be useful for studying other spinal cord pathologies such as glioblastomas, meningitis, or the cellular physiology of the normal spinal cord. For our method, vertebral stabilization was achieved by building an exoskeleton around four .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bone removal drill	World Precision Instruments	IDEAL MICRO-DRILL	Also sold by Harvard Apparatus. Any high quality surgical bone drill will suffice.
Drill burr (#1/4 or 1/2 Carbide Round Burr)	World Precision Instruments	501860 (#1/4) 501860 (#1/2)	Also sold by Harvard Apparatus
Staples	Lyreco	5.002.567	Crown = 8 mm Leg = 4 mm
Paperclip			Any standard 25 mm paperclip
Needle drivers	Fine Science Tools	12003-15
Tissue scissors	Fine Science Tools	14028-10
Adson forceps	Fine Science Tools	11027-12	1 x 2 teeth
Blunt forceps	Fine Science Tools	11293-00
Spinal forks	Harvard Apparatus	724813
Dental cement	GACD	12-565 & 12-568
Silicone elastomer	World Precision Instruments	KWIK-SIL	Also avaiable through Coherent Scientific and Fisher Scientific
Cyanoacrylate	Eleco-EFD	Cyanolit 201
Microscope	Zeiss	7MP	Our microscope is in an upright configuration and is equipped with 5 NDD detectors.
Objective lens	Zeiss	W Plan-Apochromat 20x/1.0 DIC M27 75mm	Working distance = 1.8 mm