Name: determining immune system suppression versus cns protection for pharmacological interventions in autoimmune demyelination
Uploaded: 2016-09-12T14:00:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination at JoVE.com

This work was funded by NINDS P30-NS069324, The National Multiple Sclerosis SocietyRG 4587-A-1, The Civitan International Research Foundation, The Mike L. Jezdimir Transverse Myelitis Foundation, The University of Alabama Health Services Foundation - General Endowment Fund, The National Science Foundation 1355183, and T32 AI007051 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

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The authors have nothing to disclose.

Patients with MS continue to experience disease relapses while taking drugs that attenuate T cell activation and/or infiltration into the CNS, warranting the development of treatment options that directly protect the CNS. EAE has classically been used to model the symptoms of MS and can be a powerful tool when studying the nature of interactions between the immune system and CNS in vivo. Using timing of treatment considerations in EAE, e.g., before or after initiation of disease, in conjunction with examining immune cell infiltration in the CNS and proliferation and activation in the periphery, it is possible to delineate the effects of treatments on both the immune system and the CNS.
While EAE in the C57BL/6 mouse is more widely utilized, EAE in the SJL mouse may be more representative of the majority of MS cases, as these mice have a relapsing-remitting phenotype and infiltration of immune cells in the parenchyma of the brain 10. SJL mice have clear recovery during remission as well, making it possible to begin treatment after the disease has presented but during times of reduced inflammation. It is important to consider that SJL mice do not always relapse and remit in synchrony, resulting in potentially large variability when results are pooled. Therefore, some researchers may opt to show representative results for clinical scores from one animal while taking mice for FACS analysis and histology at individualized points in disease progression.
Considering when manipulations are made to EAE mice can assist in the determination of how a treatment affects the immune system or CNS. There are many options for when treatment begins, each with its own connotation for whether immune cells have entered the CNS and how they may be interacting with the CNS. Treatment before onset of symptoms implies that immune cells have not yet entered or caused damage to the CNS. Treatment after onset of symptoms implies that immune cells have entered the CNS and have caused some damage. Using SJL mice, treatment can also begin during a relapse, where immune cells are actively infiltrating and causing inflammation, or during remission, where immune cells may be less prevalent in the CNS with less inflammation. Initial hypotheses regarding how treatments affect the CNS and immune system can be made when considering where immune cells are in the pathological process during treatment.
There are a number of ways in which treatments can affect immune cells and the CNS, each with the end result of reducing severity of EAE symptoms. Therefore, it is necessary to use flow cytometric analysis and immunohistochemistry to look at how immune cells are affected in the periphery and CNS, whether immune cells have entered the CNS, and how the CNS reacts to treatment. While flow cytometric analysis of the spinal cord can determine how many cells have entered the CNS at a given time, one cannot determine that this effect is due to reduced immune cell trafficking unless proliferation of immune cells is unaffected in the spleen. It is therefore necessary to analyze both peripheral and CNS tissue and determine what results mean mechanistically when both tissues are compared. It is also possible for immune cell activity profiles to be altered by treatment, for example having a switch in a pathogenic helper T cell-heavy profile to a regulatory T cell-heavy profile. Looking at markers for different cell types and comparing percent expression between treated and untreated animals is therefore also an important consideration. An emerging concept in MS research suggests that B cells play an important role in autoimmune demyelination. This is based on studies showing that B cells are necessary for the reactivation of T cells 20. This concept is supported by the success of treatments such as rituximab, an antibody against CD20 expressed on the surface of B cells 21,22. As demonstrated by the success of the monoclonal antibody ocrelizumab in clinical trials, drugs targeting different epitopes of CD20 may improve the efficacy of B cell-targeted therapeutics 23.
One limitation of the techniques presented here is that it is possible for immune cells to enter the CNS but be unable to travel in the parenchyma. Immunohistochemistry can be used to detect perivascular cuffing of immune cells and evaluate distance traveled in the parenchyma between treated and untreated animals. Another potential limitation involves the effects of the microbiome on EAE pathogenesis. Commensal gut microbiota can heavily influence disease pathogenesis 24; therefore, mice housed in different colonies and even in different cages can have vast differences in disease severity. Accordingly, it is always preferable where possible to use littermate controls raised in the same cage for experiments involving EAE. A final note is that if it is experimentally desirable to eliminate the effects of immune cell proliferative changes in the periphery, it may be possible to do so using passive transfer induction rather than the active induction described in this protocol.
Further confirmation for neuroprotection can be accomplished using a co-culture system 11 to test specific mechanisms of cell death or through the use of conditional knockout mice which allows for deletion of proteins selectively on a cell type. Furthermore, to extend the exploration of pharmacological agents that are neuroprotective, markers of axonal transection and neuronal death should be included. Another area of importance is remyelination. Injured axons are unable to remyelinate lending further support that neuroprotective therapies should be an important part of remyelination therapies. Additionally, unmyelinated axons are more vulnerable to injury than myelinated axons. This suggests that when an axon becomes demyelinated therapeutic interventions that promote timely remyelination will prevent axonal injury. To explore these avenues, other in vivo models for demyelination and remyelination may be used (i.e., cuprizone and lysolecithin). The method described herein focused on assessing neuroprotection by quantifying myelin loss. For the evaluation of remyelination the number of progenitor cells as well as their ability to proliferate and mature would also be important to investigate. With the mention of these alternative models, one must also consider different models of encephalitis that are virally mediated. There are two well-characterized RNA viral models that produce myelin loss: one is Theiler&#39;s murine encephalomyelitis, a non-enveloped Picornaviridae virus, and the other is mouse hepatitis virus, a member of the Coronaviridae virus family 25,26.
EAE is a valuable tool for studies of how manipulations or treatments affect the immune system and the CNS in vivo. The protocol described here can help determine where treatments are affecting the disease process, whether it be in the periphery, at the blood-brain barrier, or in the CNS. No current treatments for MS cure the disease and patients often experience decline over time. Similarly, other diseases involving immune cell infiltration into the CNS and degradation of myelin, including acute disseminated encephalomyelitis, transverse myelitis, and neuromyelitis optica, lack treatments that protect the CNS as it is directly under attack by infiltrating immune cells. Taking into consideration the timing of treatment and using flow cytometric analysis of the spleen and spinal cord in conjunction with immunohistochemistry of the CNS to assess inflammation and damage will allow for mechanistic determinations to be made regarding treatments.

Multiple sclerosis (MS) is characterized by inflammatory lesions predominantly in white matter regions of the brain early in disease. After long-term progression, gray matter atrophy is detected by MRI imaging and marks the neurodegenerative phase of the disease. Reactive gliosis, demyelination, and axonal damage in the white matter are attributed to CNS-infiltrating immune cells. None of the treatments currently used in MS reverse or directly prevent neurodegeneration in the CNS - instead, they reduce inflammation by attenuating T cell activation and/or infiltration into the CNS. Because there is no cure for MS and patients using current treatments continue to experience disease progression, discoveries of drugs that prevent demyelination and neuronal loss are critically important. However, differentiating between effects on immune cells and those on the CNS can be difficult experimentally, as the outcome - i.e., reduced damage to the CNS - looks the same regardless of the mechanisms through which it occurs. Therefore, assessment of CNS protection must be partnered with assessments of CNS-infiltrating immune cells and proliferation of immune cells in the periphery to determine how pharmacological agents affect disease mechanisms.
Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of autoimmune inflammatory disorders that was directly responsible for the discovery of drugs currently used to treat MS 1-4. Mice are often used for EAE, with C57BL/6 mice being a popular strain based on the availability of genetic variants. C57BL/6 mice induced with EAE exhibit chronic disease progression with onset around day 10 post-induction. Infiltration of the spinal cord parenchyma and cerebellum are characteristic of the histopathology of these animals, with absence of infiltration in the cortical parenchyma 5. Additionally, cortical lesions and demyelination in the brain are hallmarks of the disease 6-9, which are relatively absent in C57BL/6 mice. Therefore, it may be preferable when possible to use SJL mice, which have relapsing-remitting disease and lesions found in both the brain and spinal cord that appear similar to those in MS 10.
Treatment cannot be classified as neuroprotective if immune cells never reach the CNS. Therefore, this protocol makes use of flow cytometric analysis of brains, spinal cords, and spleens from EAE mice to determine effects of treatment on immune cell infiltration into the CNS and proliferation of immune cells in the periphery, as previously demonstrated 11. Immunohistochemical analyses of CNS tissue to determine extent and nature of neuroprotection is also described. Combining these methods allows for the determination of whether immune cells were activated and proliferated in the periphery, whether immune cells entered the CNS, and whether the CNS was protected from inflammation or damage. If neuroprotective effects are suspected despite effects on the immune system, experimenters can alter treatment start times after immune cell infiltration into the CNS has occurred.
Here, we present a protocol using two different models of active EAE, a T cell-mediated animal model of MS, and flow cytometry analysis combined with immunohistochemistry at various time-points during the disease to determine the efficacy of experimental therapies on different aspects of MS pathogenesis. This method will assist researchers in differentiating between effects on immune cell proliferation and infiltration versus CNS protection, making it easier to narrow down how drugs act on disease pathogenesis.

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determining immune system suppression versus cns protection for pharmacological interventions in autoimmune demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

Here, we used two models of EAE to understand if a pharmacological agent provides CNS protection by either attenuating CNS-infiltrating T cells or preventing myelin and axonal injury during the onslaught of inflammatory immune cell infiltration. To determine if a therapeutic agent prevents immune cell infiltration into the spinal cord, the C57BL/6 mouse model of chronic EAE is used where immune cell infiltration and disease pathology is predominantly located in the spinal cord (Figure 1A). To determine if a therapeutic drug provides CNS protection during the intrusion of immune cells into the CNS, the SJL animal model of relapsing-remitting EAE is used, which demonstrates disease pathology in both the brain and spinal cord (Figure 1B).
Clinical Assessments
Relevant clinical assessments are made according to the following rubric for typical (Figure 1C) or atypical (Figure 1D) EAE. For typical clinical disease, a score of 0 is no abnormal behavior. When picked up by the base of the tail, the tail may rotate quickly (much like a helicopter rotor) and the hind legs will spread apart. A clinical score of 1 is a partially limp tail, which may be determined by lifting the mouse by the base of the tail. The normal helicopter-like rotating may be weakened or absent, and part of the tail may be completely limp. A helpful way to determine extent of tail paralysis is to run one&#39;s finger up the length of the tail, as an unparalyzed tail will usually curl around the finger while a partially paralyzed tail will be unable to do so. A clinical score of 2 represents a completely paralyzed tail. No movement of the tail occurs at all when picking the mouse up at the base of the tail. A clinical score of 3 represents partial hind limb paralysis. Determination of this score requires that the mouse be free to move on a flat surface. If one hind limb is dragging as the mouse moves forward, or if one or both hind limbs appear to be partially paralyzed, a score of 3 may be given. A clinical score of 4 represents complete hind limb paralysis. With this score, a mouse will be unable to move its hind limbs and will drag itself forward using its front limbs. A clinical score of 5 represents a moribund mouse, or a mouse with difficulty moving itself across its cage or breathing. If a mouse cannot drag itself along the bottom of the cage or if its breathing is labored, the mouse should be humanely euthanized. A clinical score of 6 represents a mouse found dead in its cage. A score of 6 is unusual and causes of death other than EAE should be investigated.
Atypical clinical disease may or may not be accompanied by paralysis. It may be necessary to include two separate scoring systems if a mouse presents with atypical disease plus typical symptoms. A score of 0 is no abnormal behavior, as with the typical scoring system. A clinical score of 1 represents a slight head turn or tilt while the mouse is walking. This may be determined by allowing the mouse to walk forward and observing a constant left or right directionality to its movement. A clinical score of 2 represents a more pronounced head turn and poor righting ability. As with an atypical score of 1, the mouse has directionality to its movement and may have slight difficulty with balance. A clinical score of 3 represents an inability to walk in a straight line. The mouse will have difficulty balancing and may use the side of the cage to help right itself as it walks. A clinical score of 4 represents a mouse laying on its side, unable to walk due to balancing issues. The mouse may be able to drag itself along the bottom of the cage but may have directionality to its movement. A clinical score of 5 represents continuous rolling unless supported. A mouse that reaches this score should be humanely euthanized. A clinical score of 6 represents a mouse found dead in its cage. A score of 6 is unusual and causes of death other than EAE should be investigated.
It may be necessary to allow for &#34;in-between&#34; scores, e.g., adding 0.5 to a score if a mouse&#39;s condition changes slightly or if choosing between two scores is difficult. For example, a mouse that begins to move more slowly than its normal counterparts but displays no paralysis, or a mouse that clasps its hind feet with its front instead of splaying its legs out when picked up by the tail may be given a score of 0.5. A mouse that can only drag itself along the bottom of the cage and is only able to twitch its hind limbs periodically or when touched may be given a score of 3.5.
Assessing a Reduction in Immune Cell Infiltration
After induction of EAE in the C57BL/6 mouse model (Figure 1A, day 0), antigen presentation and proliferation of T cells in the spleen occur on days 1 - 5 followed by immune cell infiltration into the CNS around day 7. Approximately 3 to 5 days after the initial immune cell infiltration mice present with clinical scores. To assess if a therapeutic agent is blocking immune cell infiltration into the spinal cord, drugs or vehicle are introduced on day 7 after antigen presentation and proliferation in the spleens but before immune cells start to infiltrate into the spinal cord. If immune cell infiltration has been attenuated, the clinical disease course should reflect improved clinical scores during the rising phase of the disease from days 10 to 15 (Figure 2).
A reduction in immune cell infiltration would also result in diminished neuroinflammation. Reactive astrocytosis and microgliosis are considered major hallmarks for neuroinflammation. Staining for astrocytes with GFAP and microglia with Iba-1 can then be used to assess changes in mean area fraction staining to quantify neuroinflammation (Figure 3).
To determine if immune cell infiltration is reduced, the spinal cords are removed and processed for flow cytometry analysis at the peak of disease (Figure 1A, approximately day 18). This ensures that the largest number of immune cells have entered into the spinal cord. Entrance of T cells into the CNS is considered the initiating inflammatory event and both Th1 and Th17 cells are found in animal models of EAE as well as MS patients. Taken together, flow cytometric analysis should include assessment of both types of pathogenic T cells. Furthermore, Tregs are well-characterized suppressor T cells that dampen disease. Therefore, the percentage of Tregs from a total CD4+ population must be evaluated compared to the percentage of effector T cell populations. This will reveal if an overall reduction in T cell infiltration has occurred or if there is a skewing of T cell phenotypes in the CNS. Representative dot plots (Figure 4A) demonstrate a reduction in overall number of CD4+ infiltrating T cells in spinal cords from drug-treated mice compared with spinal cords from vehicle-treated mice (numbers in upper right quadrants). To evaluate Th1, Th17, and Treg cells the following signature proteins are evaluated: IFN-&#947;+, IL-17+, and Foxp3+, respectively and should be reduced (Figure 4A). Statistical analysis should be performed on CD4+, IFN-&#947;+, IL-17+, and Foxp3+ cell numbers to demonstrate a significant reduction (Figure 4B). To rule out a skewing of T cell subsets, statistical evaluation of the proportion of IFN-&#947;+ IL-17+, IFN-&#947;+ IL-17-, IL-17+ IFN-&#947;-, and Foxp3+ cells is performed (Figure 4C).
To eliminate the possibility that a reduction in CNS-infiltrating T cells is a consequence of inhibiting proliferation, activation, and differentiation in the periphery, the number of actively proliferating T cells in addition to the proportion of T cell subtypes needs to be evaluated. No change in the percentage of CD4+, IFN-&#947;+, IL-17+, or Foxp3+ should be found if activation and differentiation are unaffected (Figure 5A). Furthermore, no change in Ki67+ CD4+ cells should be found if proliferation is unaffected (Figure 5B). Drug treatments are introduced on day 7 or later to avoid altering initial antigen presentation and T cell activation in the periphery. However, in genetic models proteins are often deleted constitutively during embryogenesis or induced before induction of EAE making splenocyte assessment of high importance.
Assessing CNS Protection
To demonstrate if a particular therapeutic agent modulates disease pathology in the CNS after immune cell infiltration, drug interventions should be administered during the first peak in clinical disease scoring. The SJL model of EAE is advantageous for these experiments since these mice exhibit a relapsing-remitting phenotype. If a drug treatment prevents myelin-axon degeneration, an improvement in clinical scores will be observed (Figure 6). Pathological assessment of myelin must corroborate a reduction in myelin damage consistent with improved clinical scores. To quantitatively evaluate myelin integrity, DAB staining of myelin basic protein (MBP) is performed, followed by statistical analysis of the optical density for this staining (Figure 7). To further substantiate that neuroinflammation is sustained or decreased by therapeutic interventions, reactive gliosis can be assessed by measuring mean fraction area for reactive gliosis as described above (Figure 3). To corroborate that a therapeutic intervention is directly protecting the CNS without immunomodulatory effects, attenuation of immune cell infiltration into the CNS and proliferation in the spleens must be discounted. To address this, methods for brain and spinal cord assessment of immune cell infiltration and assessment of peripheral T cell proliferation and activation should be performed as described above (Figures 4 and 5). Taken together, therapeutic agents that block cell injury in the CNS with no evidence of a reduction in CNS-infiltrating T cells or proliferation of T cells in the periphery are CNS-protective treatments.
<img alt="Figure 1" src="/files/ftp_upload/54348/54348fig1.jpg" /> 
Figure 1. Representative Results of Clinical Scores from EAE in C57BL/6 and SJL Mice. (A) Clinical scores (mean &#177; SEM) of C57BL/6 mice (n = 10) induced with MOG35-55 to produce EAE with chronic disease. (B) Clinical scores (mean &#177; SEM) of SJL mice (n = 3) induced with PLP139-151 to produce EAE with relapsing-remitting disease. (C) The clinical scoring rubric used to track typical disease progression in EAE mice. (D) The clinical scoring rubric used to track atypical disease progression in EAE mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54348/54348fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54348/54348fig2.jpg" /> 
Figure 2. Pharmacological Treatment prior to Immune Cell Infiltration in C57BL/6 mice with EAE. Clinical scores (mean &#177; SEM) of C57BL/6 mice treated with PBS (n = 20) or SAS (n = 19) from day 7 postimmunization with MOG35-55. Data are from three pooled independent experiments. Statistical difference was determined using a nonparametric two-tailed Mann-Whitney U test, *p &#60; 0.05. Re-print with permission from (11).
<img alt="Figure 1" src="/files/ftp_upload/54348/54348fig3.jpg" /> 
Figure 3. Immunofluorescent Staining and Quantification of Reactive Gliosis in Spinal Cords of Control, EAE, and Treated C57BL/6 Mice. (A) Fluorescent labeling for GFAP (astrocytes) and Iba-1 (microglia) in the spinal cords of control (unimmunized) mice (left panels) and EAE mice treated with PBS (middle panels) or SAS (right panels). Scale bar = 100 &#181;m. Quantification of staining was determined using the area fraction technique to measure percent immunopositive area for GFAP (B) and Iba-1 (C). Mean &#177; SEM, n = 3 control, n = 3 SAS-treated, or n = 4 PBS-treated mice, 6 sections per mouse. Statistical differences were determined using a one-way ANOVA, *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001. Re-print with permission from (11). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54348/54348fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54348/54348fig4.jpg" /> 
Figure 4. FACS Analysis of EAE C57BL/6 Mouse Spinal Cords Demonstrating Reduced T cell Infiltration in Treated Mice. C57BL/6 mice were treated with SAS or PBS, beginning 7 d postinduction of EAE. Spinal cords were obtained on day 15. (A) Representative dot plots show Th1 (IFN-&#947;+/IL-17-) and Th17 (IFN-&#947;-/IL-17+) cells in CD4+ gate (upper panels) and T regulatory cells (Foxp3+) (lower panels). Dot plots show percentages in upper right quadrant. (B) Absolute numbers of CD4+ cells as well as IFN-&#947;+, IL-17A+, and Foxp3+ cells were statistically analyzed. (C) The change in percentage of T cell populations between SAS- and PBS-treated EAE mice was also examined. Mean &#177; SEM, n = 10 for PBS treated, and n = 9 for SAS treated from two independent experiments. Two-tailed t test was used for all bar graphs. **p &#60; 0.01. Re-print with permission from (11). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54348/54348fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54348/54348fig5.jpg" /> 
Figure 5. FACS Analysis of EAE C57BL/6 Mouse Spleens Demonstrating Equivalent T cell Expression Profiles and Proliferation in Treated and Untreated Mice. Spleens from PBS- and SAS-treated mice were analyzed 15 d postinduction of EAE. (A) The percentage of CD4+ T cells, Th1 (IFN-&#947;+/IL-17-), Th17 (IFN-&#947;-/IL-17+), and T regulatory cells (Foxp3+) in spleens from PBS-treated (n = 10) and SAS-treated (n = 9) mice from two independent experiments. (B, left panel) The percentage of Ki-67+ cells in the CD4+ population from naive spleens (n = 4) as well as from PBS- (n = 5) and SAS-treated mice (n = 5) induced with EAE. A one-way ANOVA test demonstrated statistical significance between the proportion of Ki-67+ cells from naive spleens compared with either PBS- or SAS-treated EAE spleens. No significance was observed between PBS- and SAS-treated EAE spleens. (B, right panel) Representative dot plots; numbers indicate proportion of proliferation. Dot plots show percentages. Bar graphs represent two-tailed t test, ***p &#60; 0.001. Re-print with permission from (11). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54348/54348fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54348/54348fig6.jpg" /> 
Figure 6. Pharmacological Treatment after Immune Cell Infiltration in SJL Mice with EAE. Clinical scores (mean &#177; SEM) of SJL mice treated with PBS (n = 8) or SAS (n = 8) from day 24 postimmunization (dashed line) with PLP139-151. Data are mean &#177; SEM of clinical scores. Statistical difference was determined using a nonparametric two-tailed Mann-Whitney U test, ***p &#60; 0.001. Top line represents values used for statistical analysis. Re-print with permission from (11).
<img alt="Figure 1" src="/files/ftp_upload/54348/54348fig7.jpg" /> 
Figure 7. Quantification of MBP Staining using Optical Density. (A) Representative staining of MBP in thoracic spinal cord from an unspecified genetic knockout mouse compared to littermate control C57BL/6 mouse induced with EAE. Bracket indicates representative area of reduced MBP staining indicating demyelination. (B) MBP staining of thoracic spinal cord from an unspecified genetic knockout C57BL/6 mouse. (C) Unspecified genetic knockout mice induced with EAE (KO; n = 6 mice, 2 - 4 lumbar and thoracic sections per animal) exhibit a higher optical density (OD) of MBP staining in the spinal cord than wildtype (WT; n = 3 mice, 2 - 4 lumbar and thoracic sections per animal) mice induced with EAE. Statistically analyzed using a two-tailed t test, *p &#60; 0.05. Error bars represent SEM. Scale bar 100 &#181;m.

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Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

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12.1K Views.  University of Alabama at Birmingham. This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

Video: Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Histological Staining of Spinal Cord for Assessing EAE Disease Severity

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.

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

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 ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Neuroscience

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

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