Spinal Cord Homogenate (SCH)/CFA-Induced EAE in Biozzi Mice

Typical Results | Disease Development | Treatment Regimens | Tissue Collection & Analysis Options | See Also

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Biozzi mice are immunized with spinal cord homogenate (SCH) in complete Freund's adjuvant (CFA) to induce EAE. This model recapitulates the progression from relapsing-remitting EAE to secondary progressive EAE often seen in human patients with multiple sclerosis.

During the relapsing-remitting phase of EAE, treatment with FTY720 (fingolimod, Gilenya) reduces disease severity (FTY720 prevents egress of lymphocytes from peripheral lymph nodes).

However, during the secondary progressive phase, FTY720 does not significantly reduce disease severity, indicating that this phase of EAE is not dependent on additional lymphocyte infiltration into the CNS. Therefore, this phase of EAE resembles inactive secondary progressive multiple sclerosis in human patients.

Treatment with BTK inhibitors, which disrupt B cell development and reduce myeloid cell activation, significantly ameliorate secondary progressive EAE in Biozzi mice. See data below and our 2023 publication in Acta Neuropathologica Communications.

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Typical results - Therapeutic treatment (from 2nd day of disease)

Day 0 was the day of immunization. Mice were treated with vehicle or FTY720 daily from the 2nd day of disease through Day 45 after immunization. The relapse period in this experiment was determined to start on Day 27, when the first mouse had a sign of relapse.

Therapeutic FTY720 treatment typical scores
Therapeutic FTY720 treatment typical weight change

Readouts at enrollment (mean ± SD)

Treatment Day of onset Score at enrollment
(2nd day of disease)		 Maximum score
through Day 26
Vehicle 15.1 ± 1.4 2.50 ± 0.48 2.83 ± 0.44
FTY720 14.8 ± 1.5 2.64 ± 0.39 2.95 ± 0.27

Readouts of efficacy (mean ± SD)

Treatment Relapse period
maximum score
(Days 27 to 46)1		 p value End score1 p value Relative end
body weight
(%)2		 p value
Vehicle 3.20 ± 1.14 – 2.55 ± 1.36 – 99.5 ± 12.0 –
FTY720 1.35 ± 0.24 <0.001 0.90 ± 0.21 <0.001 107.6 ± 5.1 0.066

1 Significance for relapse period maximum score and end score was calculated using two-tailed Mann-Whitney tests.
2 Percent relative end body weight is relative to body weight on Day −1 (day before immunization). Significance for relative end body weight (%) was calculated using a two-tailed unpaired t test.

Typical results - Late therapeutic treatment (from Day 49 post-immunization)

Mice were treated with vehicle, FTY720, or ibrutinib (a BTK inhibitor) daily from Day 49 through Day 82 or Day 83.

Results below are from two independent experiments.

Late therapeutic FTY720 treatment typical scores
Late therapeutic FTY720 treatment typical weight change
Late therapeutic ibrutinib treatment typical scores
Late therapeutic ibrutinib treatment typical weight change

Readouts at enrollment (mean ± SD)

Treatment Day of onset Score at onset Score at
enrollment
(Day 49)		 Maximum score
through Day 49
Vehicle for FTY720 14.8 ± 1.7 1.50 ± 0.52 1.75 ± 0.63 3.03 ± 0.34
FTY720 14.9 ± 1.4 1.50 ± 0.71 1.77 ± 0.70 3.13 ± 0.30
Vehicle for ibrutinib 14.9 ± 2.0 1.11 ± 0.72 2.33 ± 0.42 3.25 ± 0.26
Ibrutinib 14.7 ± 1.7 1.25 ± 0.69 2.31 ± 0.49 3.28 ± 0.31

Readouts of efficacy (mean ± SD)

Treatment Efficacy period
maximum score1,2		 p value End score2 p value Relative end
body weight
(%)3		 p value
Vehicle for FTY720 2.16 ± 0.72 – 1.97 ± 0.79 – 109.2 ± 13.7 –
FTY720 1.79 ± 0.58 0.099 1.54 ± 0.63 0.125 112.9 ± 13.2 0.467
Vehicle for ibrutinib 2.86 ± 0.45 – 2.56 ± 0.48 – 94.5 ± 13.4 –
Ibrutinib 2.47 ± 0.44 0.023 2.14 ± 0.64 0.042 101.9 ± 11.8 0.091

Each treatment group was compared to its respective Vehicle group.
1 The "efficacy period" is from 15 days after treatment starts (Day 64) through study termination (Day 83 or Day 84), as treatments require some time to show effects.
2 Significance for efficacy period maximum score and end score was calculated using two-tailed Mann-Whitney tests.
3 Percent relative end body weight is relative to body weight on Day -1 or Day 0. Significance for relative end body weight (%) was calculated using a two-tailed unpaired t test.

Disease development

EAE develops in Biozzi mice after immunization with an emulsion of SCH in CFA. SCH contains antigens which initiate expansion and differentiation of CNS-specific autoimmune T cells.

Over 90% of mice experience EAE onset between Days 12 and 17 after immunization.

Most mice experience the first relapse starting between Days 28 and 35 after immunization, with over 80% of mice experiencing the first relapse by Day 49 post-immunization. Between 25 and 50% of mice experience additional relapses after Day 49. However, clinical scores are not significantly reduced by treatment with FTY720 compared to vehicle, indicating that pathology beyond Day 49 is primarily driven by mechanisms other than CNS-infiltrating lymphocytes. The remaining mice experience stabilized or slowly worsening EAE after Day 49.

Direct EAE induction in Biozzi mice

Direct EAE induction in Biozzi mice

Treatment regimens

This model can be used with any of the standard EAE treatment regimens below, but is most often used with late therapeutic treatment.

Prophylactic treatment

In prophylactic studies mice are treated from the time of immunization and are most often followed for 28 days.

Overall treatment efficacy is indicated by both delayed onset of EAE and reduced maximum severity (vs. negative controls). Of these, mean maximum score (MMS) is more important - reduced MMS indicates an overall reduction in EAE severity. Median time to disease onset is usually the most sensitive measure of compound efficacy because even small changes in immune responses can delay disease onset.

In addition to FTY720 (fingolimod, Gilenya), ibrutinib (Imbruvica), glatiramer acetate (Copaxone), and dimethyl fumarate (BG-12, Tecfidera) are efficacious when dosed prophylactically.

Semi-therapeutic treatment

Semi-therapeutic treatment starts after mice have been immunized, but before all mice are sick. Usually treatment starts 10 to 11 days after immunization, or when 10 to 20% of mice have become sick.

Therapeutic treatment

Therapeutic treatment specifically demonstrates whether a compound will reverse the course of disease or improve recovery. In therapeutic treatment mice are usually enrolled into treatment groups one at a time, at EAE onset for each mouse. Groups are balanced for similar time of EAE onset and severity at enrollment - this gives very uniform groups and makes the model more sensitive to compound efficacy. We often recommend this stringent treatment regimen as a follow-up to prophylactic studies, to establish compound efficacy after disease onset.

Late therapeutic treatment

Late therapeutic treatment is similar to therapeutic treatment, except that treatment begins either a fixed number of days after each mouse shows the first signs of disease, or a fixed period after immunization. Most often, this treatment regimen is used to evaluate neuroregenerative effects.

In addition to day of EAE onset and onset score, groups are also balanced for maximum score before enrollment.

In Biozzi mice, secondary progressive EAE begins around Day 49 after immunization. During this disease phase, treatments that inhibit lymphocyte infiltration into the central nervous system are not expected to be efficacious.

Tissue collection and end-of-study analysis

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