Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Summary

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Abstract

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.

Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). ¹¹C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 µL) and corpus callosum (3 µL) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).

Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Introduction

Multiple sclerosis (MS) is a neuroinflammatory disease that affects the central nervous system, characterized by inflammation, demyelination, and axonal loss¹. The prognosis of this disease is variable even with advances in treatment, and it is one of the most common causes of neurological deficits in young people¹. The diagnosis of MS is based on the criteria of clinical manifestation and visualization of characteristic lesions by magnetic resonance imaging (MRI)^2,3.

Positron emission tomography (PET) can be a useful tool for in v....

Protocol

All procedures were conducted in accordance with the guidelines of the National Council for the control of Animal Experimentation (CONCEA, Brazil) and were approved by the Ethics Committee for Animal Research of the Medical School of the University of Sao Paulo (CEUA-FMUSP, Brazil - protocol number: 25/15).

NOTE: In this protocol, we show how to induce a lysolecithin rat model of multiple sclerosis and how to acquire and analyze the myelin PET images.

1. Lysolecithin solution preparation

1. Weigh lysolecithin (L-α-Lysophosphatidylcholine from egg yolk) on an analytical scale using a conic plastic tu....

Results

Figure 1 shows illustrative ¹¹C-PIB PET images with myelin changes over time. In the baseline scan, no differences can be seen in myelin content (i.e., no demyelination is present). In the 1-week time-point image, it is possible to see the focal demyelinated lesion (in the right hemisphere) as indicated by the white arrow. Images are presented in the 3 anatomical planes (coronal, axial, and sagittal) and it is possible to identify the demyelinated lesion in all of them. The 1.......

Discussion

The biggest advantage of using the lysolecithin model to study multiple sclerosis is the fast timeline for demyelination (about 1 week) and remyelination (about 4 weeks) to occur¹⁴. This model can also be induced in mice¹⁵, however, induction in rats is more advantageous for in vivo PET imaging due to the larger size of the rat brain compared to mice.

The first step of the induction model is to be extremely cautious. This model was valid.......

Materials

Name	Company	Catalog Number	Comments
Analytical Balance	Marte	AUWZZOD	max: 220 g- min: 1 mg
Anestesia vaporizer	Nanitech	15800
Beta-cube	Molecubes
Bulldog clamp	Stoelting	5212043P
clorexidine	Rioquimica		0.5%/100 mL
Cotton swabs	johnson e johnson
Dose calibrator	Capintech
Drill	Kinzo powertools	352901	Model Q0M-DC3C
Eppendorf tube	Eppendorf	30125150	1.5 mL
Eye lubricant	ADVFARMA	30049099	vaseline 15 g (pharmaceutical purity)
Fine forceps	Stoelting	52102-38P
Gloves	Descarpack	212101	6.5 size
Heating pad	Softhear
Injection Syringe	Hamilton	80314	10µ, 32ga, model 701
Insuline syringe	BD	328328	1 mL insulin syringes with needle
Isoflurane	Cristália	410525	100 mL , concentration 1 mL/1 mL
Ketoprofen or other analgesic	Sanofi		100 mg/2 mL
lidocaine	Hipolabor	1.1343.0102.001-5	2%/20mL
L-α-Lysophosphatidylcholine from egg yolk	Sigma-aldrich	L-4129	25 mg - ≥99%, Type I, powder
Needle holder	Stoelting	5212290P
Oxygen	White Martins	7782-44-7	Compressed gas
PMOD software	PMOD technologies	Version 4.1	module fuse it
Rat anesthesia mask	KOPF	Model 906
Saline	Farmace	0543325/ 14-8	0.9% sodium chloride for injection, 10 mL
Scapel blades	Stoelting	52173-10
Scapel handles	Stoelting	52171P
Scissor	Stoelting	52136-50P
Semi-analytical Balance	Quimis	BK-3000	max:3,100 g; min:0.2 g
shaver	Mega profissional		AT200 model
Stereotactic Apparatus	KOPF	Nodel 900
Universal holder with needle support	KOPF	Model 1772-F1	Hamilton support for 5 and 10 µL