Induction of Experimental Autoimmune Encephalomyelitis (EAE) in cynomolgus monkey: a valuable model of auto-immune demyelinating diseases
Julien Flament1,2, Claire-Maëlle Fovet1,3, Lev Stimmer1,3, Philippe Hantraye1,2,4, and Ché Serguera1,2

1CEA/DSV/I2BM/MIRCen, Fontenay-aux-Roses, France, 2INSERM UMS 27, Fontenay-aux-Roses, France, 3INSERM UMR 1169, Fontenay-aux-Roses, France, 4CNRS Université Paris-Saclay UMR 9199, Fontenay-aux-Roses, France

Synopsis

Acquired demyelinating diseases are a major cause of neurological disabilities. If Experimental Autoimmune Encephalomyelitis (EAE) model has been widely used in rodents, it does not recapitulate disease variability observed in humans. We propose for the first time a primate model of EAE without immunomodulatory treatment in Macaca fascicularis which exhibited a more developed immune system than rodents. All monkeys developed MRI visible lesions that were significantly correlated to clinical signs onset. Our longitudinal follow up allows a precise monitoring of lesions and may offer the opportunity to better understand biological and physiological processes underlying the pathology of demyelinating diseases.

Target audience

This work should be of interest to all persons using animal models to study Multiple Sclerosis and Acquired Demyelinating Diseases.

Purpose

Acquired demyelinating diseases are a major cause of neurological disabilities with an incidence of about 7/100 0001 in western Europe. Demyelinating diseases are mainly monophasic with one attack and no relapse, such as acute demyelinating encephalomyelitis (ADEM), but 30% evolve in multiphasic pathologies such as multiple sclerosis (MS). Experimental Autoimmune Encephalomyelitis (EAE) model has been widely used as a generic model of MS, especially in rodents2. In contrast to the human immune system, laboratory germ-free or gnotobiotic mice exhibit an immature and naive immune system. Consequently, the relevance of this simplistic EAE rodent model has been often criticized. Here, we propose a new Non-Human Primate (NHP) model of EAE in Macaca fascicularis3 which exhibite a more developed immune system and are biologically close to the human.

Material and Methods

Animal model: 8 adult monkeys were induced with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) formulated in incomplete Freund’s adjuvant (IFA)4. Immunogen was injected intradermally into the dorsal skin in 6 spots of 100μl each. Immunizations were repeated every 28 days until clinical signs.

Clinical score: Monkeys were observed every day and clinical signs were scored using a semi-quantitative scale from 0 to 5 (with respect to signs severity)5. Endpoints based on maximum cumulative discomfort have been defined as the end of the experimentation.

MRI: Images were acquired on a 7T Agilent magnet. Turbo-Spin-Echo sequence was used to acquire T2-weighted images (resolution=450μm2, 1.5mm thickness). FLAIR images were acquired with an inversion time of 1450ms. A rapid T1-weighted Gradient-Echo sequence was used to acquire images pre and post-gadolinium (Gd) contrast agent intraveinous injection (0.1 mmolL/kg). T2 maps were obtained using Multi-Echos-Multi-Slices sequence (10 echo times, TE1 = 15 ms and echo-spacing of 15 ms).

Results

All monkeys immunized with rhMOG/IFA developed EAE with clinical signs and brain lesions within 60 days post-immunization (dpi). We observed 70% of fulminent forms and 30% of relapsing-remitting forms. An example of clinical score time-course is shown (Fig.1a). MRI sessions and corresponding image were represented by colored arrows (Fig.1b). Clinical signs onset at 67 dpi was correlated with lesion onset (red arrow). Animal was sacrificed at 290 dpi due to high clinical signs (grade 5) and ex-vivo MRI revealed a massive demyelinated and edematous lesion through the brain (Fig.1b, orange image). Two other examples of white matter lesions are shown (Fig.2). Lesion follow-up was performed using T2-mapping (Fig.3). T2 maps were acquired before immunization (t0), and at 21 (t1), 32 (t2), 36 (t3) and 42 (t4) dpi. If lesion was visible on T2-weighted images (Fig.3, top panel, white box), it was enhanced on T2 maps (Fig.3, middle panel). Differences between T2 map at time ti and baseline T2 map were also performed for each time point. A zoom in the lesion region is shown (Fig.3, bottom panel, white box). Lesion detection was eased on difference images, especially at t3 and t4. Difference maps revealed heterogeneity with distinguishable core and rim in the lesion with various T2 values.

Discussion and Conclusion

This study proposes for the first time a monkey EAE model of demyelinating diseases without immunomodulatory treatment6. Our MRI protocol helps in discriminating nature of the lesion as on FLAIR sequence, only the central part of the lesion remained hyperintense indicating a demyelination, whereas the border was probably due to edema. Post Gd-injection images showed recently active lesions, where the blood-brain barrier (BBB) was disrupted. Our approach of longitudinal follow up of lesion based on a quantitative parameter (T2 value) associated with an individual realignment of each T2 map of each animal should allow an automated detection of lesion load without subjective bias due to misinterpretation of T2 weighted images. The development of a NHP model of EAE is a valuable tool for basic and clinical research. It offers the opportunity to better understand biological and physiological processes underlying the pathology of demyelinating diseases. Moreover, this model could constitute an appropriate tool for preclinical evaluation of new therapeutic strategies with disease follow-up using MRI.

Acknowledgements

No acknowledgement found.

References

1 Zettl U.K. et al. Autoimmun Rev 11, 167, (2012)

2 Hohlfeld R., Wekerle H. PNAS 2, 101, (2004)

3 Haanstra K.G. et al. J Neuroimmune Pharmacol 8, 5, (2013)

4 Kerlero de Rosbo N. et al. Eur J Immunol 27, 11, (1997)

5 Kerlero de Rosbo N. et al. J Neuroimmunol, 110, (2000)

6 Rose et al. Clin Immunol Immunopathol, (1987)

Figures

Figure 1: Example of clinical score time-course for one animal with MRI sessions (a, colored arrows). MR images were shown with corresponding colors (b). Lesion appearance (red arrows) was correlated to clinical signs onset.

Figure 2: Example of particular lesions observed in white matter. Lesion form and contrast were different on T2-weighted (left column), FLAIR (middle column) and post-Gd images (right column) indicating various lesion types.

Figure 3: T2-weighted images (top panel), T2 maps (middle panel) acquired before and 21, 32, 36 and 42 dpi (t0, t1, t2, t3 and t4 respectively) and difference-map between T2 map acquired at time ti (i=1,2,3 or 4) and baseline T2 map (bottom panel, white box: zoom in lesion region).



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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