paraffin embedding and histopathological scoring of demyelinating lesions and perivascular cuffs in mouse brain and spinal cord tissues

Histological Staining of Spinal Cord for Assessing EAE Disease Severity

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.

Author Spotlight: Unveiling the Pathway Linking Obesity to Autoimmune Inflammation in Multiple Sclerosis 

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

We're interested in understanding how autoimmunity and multiple sclerosis or MS gets started. This research uses a variety of approaches to study how risk factors associated with MS.Lead to the development of autoimmunity. Experimental autoimmune encephalomyelitis or EAE is a mouse model of MS that can be used to examine the effect of various risk factors on autoimmunity.We have recently discovered the biological pathway of how one MS risk factor obesity, leads to heightened autoimmune inflammation in mice. Studies in the EAE model were critical to unraveling these mechanisms. EAE disease severity is often scored on a five point clinical scale.However, this scale has limited sensitivity, and cannot distinguish whether clinical deficits are driven by inflammation or tissue damage. Histology on the spinal cord and brain is the gold standard method to assess inflammation and tissue damage in EAE. This protocol provides a simple method for measuring inflammation and tissue damage in the spinal cord and brain during EAE.Our protocol can be used alongside the clinical scale to better characterize the severity of EAE. Our protocol measures the key histological features of EAE, including immune infiltration, demyelination, axonal damage in the spinal cord, and brain inflammation. It also includes measuring neurofilament light in mouse serum, which indicates the extent of CNS injury.This comprehensive protocol provides a more detailed characterization of EAE, than using only one marker.

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

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Immunology and Infection

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