Alterations in cortical thickness and axonal density due to Experimental Autoimmune Encephalomyelitis in the Lewis Rat
J. Keiko McCreary1, Brietta Gerrard1, L. Sorina Truica1, and Gerlinde A.S. Metz1

1Neuroscience, Canadian Centre for Behavioural Neuroscience, Lethbridge, AB, Canada


The animal model of experimental autoimmune encephalomyelitis (EAE) is characterized by inflammatory lesions and demyelination which leads to axonal damage and subsequent neuronal death within the central nervous system. The effect is debilitating, resulting in loss of motor and sensory functions. Here, we investigated changes in cortical thickness using MRI, and axonal density in the corpus callosum using a neuronal tract tracer, biotinylated dextran amine, in Lewis rats induced with EAE. Our study found that EAE leads to a decrease in cortical thickness, particularly in the primary somatosensory trunk region, and axonal density in the corpus callosum.


Experimental autoimmune encephalomyelitis (EAE) is characterized by inflammatory lesions and demyelination which leads to axonal damage and subsequent neuronal death within the central nervous system. EAE is the most commonly used animal model of MS and mimics the hallmark neuropathological features of human MS1,4. Previous studies have found conflicting data for the effect of EAE on cerebral cortex volume2. In this study, we investigated changes in cortical thickness using MRI, and axonal density in the corpus callosum using a neuronal tract tracer, biotinylated dextran amine (BDA). Our hypothesis was that the disseminated loss of myelin reduces cortical thickness and axonal density in the corpus callosum.


For this study, twelve female Lewis rats (Charles River Laboratories, Quebec) were used. The animals were housed in groups of two or three under standard environmental conditions (12:12 hour light/dark cycle with lights on at 7:30 AM). Animals had access to food and water ad libitum. Experimental and surgical procedures were performed in accordance with the guidelines of the Canadian Council on Animal Care at the University of Lethbridge. In order to induce EAE, rats were immunized with guinea pig (GP) myelin basic protein (MBP) (segment 69-88: YGSLPQKSQRSQDENPVVHF) obtained from GenScript (Piscataway, NJ) for monophasic EAE 3,4. Experimental groups were (1) MBP-inoculated control (n=7; EAE) and (2) CFA injected (n=5; Control). EAE was induced by subcutaneous immunization at the base of the tail with GP MBP69-88 emulsified in Freund’s adjuvant (Difco Laboratories, BD Bioscience). Freund’s adjuvant was supplemented with 4 mg/ml heat killed Mycobacterium tuberculosis H37Ra (Difco Laboratories, BD Biosciences) to make complete Freund's adjuvant (CFA; 1 mg/ml). After onset of symptoms, all animals were injected for white matter tract tracing with 1uL of BDA in to the motor cortex. MRI was performed on a 4.7T 330 mm bore Oxford magnet (Oxford, UK) and a MR6000 (MR Solutions, UK) console, using a quadrature volume transmit-receive coil (OD=40 mm, ID=20 mm, Active Volume Length =20 mm, Morris Instruments, Ottawa). Structural T2-weighted images (SEMS: TE/TR 75/ 3000 ms, 0.23 x 0.23 x 2 mm3, slice positioning: coronal) were acquired. To ensure reproducibility between rats, the third slice was positioned to be tangential to the occipital bone. Slices positioned from Bregma 2.20 mm to Bregma -5.60 mm (Figure 1) were used for cortical thickness analysis (ImageJ, NIH). Seven measurements on the right hemisphere were obtained per slice (Figure 2A) in addition to the area of each slice. Measurements were hand drawn by a student that was blind to the group, and purpose of the experiment. Thickness coordinates were compared to Paxinos and Watson rat atlas5 to determine location of measurement. Two sets of data were collected: thickness measurements and proportional thickness derived from brain slice area. Rats were euthanized 2 weeks after symptom onset and brains were extracted for tissue processing. Data was analyzed using SPSS (v21.0, IBM, NY).


Results show cortical thinning in somatosensory regions after two weeks of EAE inoculation. Particularly, there is a significant decrease in cortical thickness on the slice corresponding to Bregma -2.56 mm in two regions (p<0.05; Figure 2). Moreover, using BDA, we found a significant decrease in axonal density as measured by integrated density in the corpus callosum after EAE (p<0.05; Figure 3). Furthermore, regression analysis indicates that the change in cortical thickness can moderately be explained by changes seen in corpus callosum (R=0.512; R2=0.262; Figure 4).

Discussion and Conclusion

These results provide insight into cortical plasticity in EAE and indicate white matter loss in the corpus callosum. Interestingly, cortical thickness was significantly decreased in the somatosensory region corresponding to the trunk of the animal. It should be noted that in this animal model, EAE also reduces mechanical nociceptive responses to von Frey hair stimulation of the hind-paw, however, the trunk region was not measured4. This region could provide a useful target area for the detection of sensation loss as this is one of the earliest symptoms in MS6. Generally, a loss of sensation in trunk regions would go unnoticed. This finding possibly offers a new strategy for the identification of predictive biomarkers of MS. Further investigations will include the measurement of whole brain cortical thickness and segmentation using automated methods by Lee and Colleagues7, along with tract density analysis in the spinal cord.


This research was supported by the Natural Sciences and Engineering Research Council of Canada (GM), and Alberta endMS (GM). JKM and BG were endMS SPRINT scholars.


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Figure 1. T2- weighted MRI images of the slices used for cortical thickness analysis and their slice positioning relative to Bregma.

Figure 2. T2-weighted MRI; 7 lines were drawn on each slice and coordinates were obtained using Paxinos and Watson rat atlas. B) Cortical thickness measurements in RSA, M2, S1, S2 and Aud are shown here. Significant differences were found in the S1 region. Values represent mean ± S.D. *P<0.05. Abbreviations: Retrosplenial agranular (RSA), Motor (M2), Somatosensory (S1, S2), Auditory (Aud).

Figure 3. 10X magnification of the corpus callosum of A) control animal and two EAE animals (B, C). D) Results show that the integrated density indicates a significant decrease in fiber density in EAE animals. Values represent mean ± SD. *P<0.05

Figure 4. Regression plot showing the significant relationship between integrated density measured in the corpus callosum and cortical thickness in the somatosensory cortex.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)