Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Summary

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Abstract

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

Introduction

Multiple sclerosis is caused by the autoimmune-mediated attack and destruction of the myelin sheath in the brain and the spinal cord¹. With an overall incidence of about 3.6 cases per 100,000 people a year in women and about 2.0 in men, MS is the second most common cause of neurological disability in young adults, after traumatic injuries^2,3. The disease pathology is contributed to by genetic and environmental factors⁴ but is still not completely understood. Autoreactive T lymphocytes enter the central nervous system and trigger an inflammatory cascade that cause....

Protocol

1. EAE Induction in SJL/J Mice

1. Mice
   1. Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = 10 mice per group.
   2. For the assessment of medication effects, administer the drug and placebo for the control group continuously via the drinking water or via food pellets starting 3 or 5 days after immunization (n = 10 per group). During the peak of the disease, administer medication or placebo with milk or 3% sugar water-soaked corn flakes.
2. Immunization material
   1. Use an EAE induction kit consisting of antigen (peptide of proteolipid p....

Results

Time Course of Bioluminescence of Optic Neuritis

The bioluminescence signal of the inflammation probe was the strongest around the eyes and occurred exclusively in EAE mice with optic neuritis. A signal occurred in neither the non-EAE mice nor the mice not injected with the inflammation probe. The signal disappeared when the mice recovered. Hence, the signal is specific for optic neuritis, and the peak of the si.......

Discussion

The present video shows techniques for bioluminescence and near-infrared fluorescence in vivo imaging of EAE in SJL/J mice. We show that bioluminescence imaging using an inflammation-sensitive probe mainly shows optic neuritis, and the quantification agrees with the clinical evaluation of EAE severity and the effects of medication. However, the bioluminescence imaging method was not able to detect inflammation of the lumbar spinal cord, which is a primary site of EAE manifestation¹⁷, like.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AngioSpark-680	Perkin Elmer, Inc., Waltham, USA	NEV10149	Imaging probe, pegylated nanoparticles, useful for imaging of blood brain barrier integrity
MMP-sense 680	Perkin Elmer, Inc., Waltham, USA	NEV10126	Imaging probe, activatable by matrix metalloproteinases, useful for imaging of inflammation
XenoLight RediJect Inflammation Probe	Perkin Elmer, Inc., Waltham, USA	760535	Imaging probe, activatable by oxidases, useful for imaging of inflammation
PLP139-151/CFA emulsion	Hooke Labs, St Lawrence, MA	EK-0123	EAE induction kit
Pertussis Toxin	Hooke Labs, St Lawrence, MA	EK-0123	EAE induction kit
IVIS Lumina Spectrum	Perkin Elmer, Inc., Waltham, USA		Bioluminescence and Infrared Imaging System
LivingImage 4.5 software	Perkin Elmer, Inc., Waltham, USA	CLS136334	IVIS analysis software
Isoflurane	Abbott Labs, Illinois, USA	26675-46-7	Anaesthetic