harvesting and fixing mouse brain and spinal cord tissues for histological scoring of experimental autoimmune encephalomyelitis

Coloration histologique de la moelle épinière pour évaluer la gravité de la maladie EAE

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for the human demyelinating disease, Multiple Sclerosis (MS)1. Classic MS inflammatory pathology, including the infiltration of IFN-&#947; (gamma) and IL-17-producing T helper cells2, the infiltration of inflammatory monocytes3, the formation of perivascular and sub-meningeal inflammatory demyelinating lesions4, and the occurrence of axon injury4 in the central nervous system (CNS), is also observed in EAE5,6,7,8,9. The similarity in immune mechanisms between EAE and MS has made EAE a suitable pre-clinical model for testing the efficacy and mechanisms of action of a number of approved immune-based therapies for MS, including natalizumab, fingolimod, dimethyl fumarate, and glatiramer acetate (reviewed in1,5). Certain EAE regimens model other aspects of progressive MS pathology beyond axonal injury, including the development of sub-meningeal inflammation in the brain, chronic demyelination, spinal cord atrophy, synapse, and neuron loss6,10,11,12. Thus, EAE has utility for screening the efficacy of neuroprotective therapies for MS.
EAE is induced in rodents in a number of ways. Active immunization is the most common induction method and involves immunization of rodents with myelin antigens (either whole proteins or peptides) emulsified in CFA supplemented with heat-killed Mycobacterium tuberculosis13. Depending on the strain of mouse, pertussis toxin (PTX) is also administered on day 0 and day 2 of immunization to increase the penetrance of disease13. EAE can also be induced by adoptively transferring myelin-specific T cells obtained from myelin/CFA-primed mice into healthy mice14&#160;or can develop spontaneously in mice that overexpress T cell receptors specific for the major myelin antigens5.
EAE disease severity and progression are commonly scored using a discrete 5-point clinical scale: 1 &#8211; tail limpness, 2 &#8211; hindlimb or foot weakness, 3 &#8211; complete paralysis in one or both hindlimbs, 4 &#8211; forelimb weakness, 5 &#8211; moribund or dead13. This clinical scoring system is sound in documenting the progression of ascending paralysis that occurs at disease onset but is less sensitive at capturing the extent of recovery from CNS inflammatory attacks. For example, both mice that ambulate with difficulty and mice that ambulate easily but exhibit foot-grasping weakness are assigned a score of 2 on the EAE scale. Scores can remain high in the post-acute phase of EAE due to the presence of permanent axon injury or loss, even despite the resolution of the inflammatory response9. There have been a variety of attempts at developing more refined scoring systems, behavioral tests, measures of hindlimb and grip strength, and infrared monitoring systems&#160;to better capture differences in clinical deficits in EAE9,16,17,18; however, these more intricate scoring measures do not distinguish the contribution of inflammation versus tissue injury to the underlying neurological deficits. Thus, the gold standard approach to score the severity of EAE is to conduct both clinical and histological scoring.
Here, a protocol is described for how to dissect and embed mouse spinal cord and brain specimens in paraffin in a way that captures the stochastic process of lesion formation that occurs in EAE. A protocol is also presented of how to stain sections with Luxol fast blue (LFB), originally created by Kluver and Barrera19, which detects myelin in the CNS. Sections are either stained with LFB alone (for demyelination analysis) or are counter-stained with hematoxylin and eosin (H&#38;E) to help visualize and score inflammatory lesions. Protocols are also provided to quantify the presence of total leukocytes (CD45), the loss of myelin, and the number of injured axons (SMI-32) in the spinal cord using commercially available antibodies, immunohistochemical (IHC) techniques, and publicly accessible software. The protocol used to quantify leukocytes in the spinal cord can also be applied to quantify leukocytes in the brain.
Histological evaluation of axonal loss and injury in the brain is comparatively more difficult than in the spinal cord since brain white matter tracts do not run in parallel to one another. The measurement of serum neurofilament light (sNF-L) has emerged as a promising biomarker for&#160;neuronal injury in MS20,21. Recent studies have extended this technology to EAE22,23,24. Here, a method is presented to measure serum neurofilament light (sNF-L) in living mice using a Small Molecule Assay (SIMOA) assay. This method requires only a small quantity of serum and can be done in live mice in just half a day, providing rapid feedback on how a tested therapy is affecting overall CNS injury. All of the methods described here can be applied to mice of any sex or strain.

Pleins feux sur l’auteur : Dévoilement de la voie reliant l’obésité à l’inflammation auto-immune dans la sclérose en plaques 

Évaluation de l’inflammation, de la démyélinisation et des lésions axonales du système nerveux central dans l’encéphalomyélite auto-immune expérimentale

Watch this Scientific Journal Video about Harvesting and Fixing Mouse Brain and Spinal Cord Tissues for Histological Scoring of Experimental Autoimmune Encephalomyelitis at JoVE.com

Harvesting and Fixing Mouse Brain and Spinal Cord Tissues for Histological Scoring of Experimental Autoimmune Encephalomyelitis  

Prélèvement et fixation de tissus du cerveau et de la moelle épinière de souris pour le pointage histologique de l’encéphalomyélite auto-immune expérimentale

Pour commencer, utilisez une pince pour saisir la peau sur le dessus de la tête d’une souris euthanasiée et faites une incision de 2,5 centimètres à l’aide de ciseaux chirurgicaux. Utilisez les doigts pour pousser latéralement la peau de la tête afin de visualiser le crâne sous-jacent. Ensuite, à l’aide de la pince Adson standard, saisissez les orbites pour stabiliser la tête.À l’aide de ciseaux fins, faites de petites cisailles dans le crâne le long de la ligne médiane de la colonne cervicale jusqu’aux bulbes olfactifs. Utilisez des pinces Adson avec des dents pour refléter le crâne ouvert afin de révéler le cerveau sous-jacent. Tenez le cerveau à l’aide de la main non dominante.Avec les ciseaux fermés dans la main dominante, retirez la moelle épinière de la colonne cervicale et poussez doucement le cerveau hors du crâne, en coupant les nerfs crâniens. Placez le cerveau dans un tube conique contenant 10 millilitres de formol tamponné neutre à 10 %. Ensuite, incisez la fourrure le long de la ligne médiane du torse de la souris, du cou à la queue.Utilisez les doigts pour pousser la peau latéralement afin de visualiser la colonne vertébrale. À l’aide de ciseaux chirurgicaux, coupez vers le bas à travers la colonne vertébrale au point d’attache du fémur à la hanche. Ensuite, coupez la paroi du corps de chaque côté de la colonne vertébrale, de la hanche au cou.Placez la colonne vertébrale contenant la moelle épinière dans le même tube contenant le cerveau et le formol.

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Research

JoVE Journal

Immunology and Infection

Harvesting and Fixing Mouse Brain and Spinal Cord Tissues for Histological Scoring of Experimental Autoimmune Encephalomyelitis

730 vues. Pour commencer, utilisez une pince pour saisir la peau sur le dessus de la tête d’une souris euthanasiée et faites une incision de 2,5 centimètres à l’aide de ciseaux chirurgicaux. Utilisez les doigts pour pousser latéralement la peau de la tête afin de visualiser le crâne sous-jacent. Ensuite, à l’aide de la pince Adson standard, saisissez les orbites pour stabiliser la tête.À l’aide de ciseaux fins, faites de petites cisailles dans le crâne le long de la ligne méd...

Vidéo: Harvesting and Fixing Mouse Brain and Spinal Cord Tissues for Histological Scoring of Experimental Autoimmune Encephalomyelitis  

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund&#39;s adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into na&#239;ve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS).
Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides&#160;feedback on the extent of overall CNS injury in live mice.

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by ...

Recherche

Immunologie et infection

Paraffin Embedding and Histopathological Scoring of Demyelinating Lesions and Perivascular Cuffs in Mouse Brain and Spinal Cord Tissues

Enrobage de paraffine et marquage histopathologique des lésions démyélinisantes et des coiffes périvasculaires dans les tissus du cerveau et de la moelle épinière de souris