Induction Of Experimental Allergic Encephalomyelitis In Swine And Study Of Spinal Cord Diffusion Tensor Imaging

BackgroundMultiple sclerosis (MS) is a chronic progressive disease of the central nervous system (CNS) which is histopathologically characterized by focal demyelinated lesions with different degrees of inflammation, axonal damage and glial scar formation. Although the hallmark of the process is demyelination, axonal pathology has been known to occur in multiple sclerosis from the earliest post-mortem descriptions of the disease. It has been widely speculated that axonal loss is the pathologic correlate of irreversible neurological impairment in MS. So far, no therapy has been proven to stop or prevent axonal injury, the pathogenesis of which is largely unknown. So a new marker to investigate underlying pathogenesis processes of MS and guide therapeutic management in more precisely is required.In humans, biopsy or autopsy are only approach to investigate underlying pathogenesis processes in MS, The data are of course limited as they give only a snapshot of the disease process .Therefore, animal models of disease are necessary to elucidate the manner in which diffusion properties reflect developmental or pathological changes. Experimental allergic encephalomyelitis (EAE) is a neurological autoimmune disease and the primary animal model for MS. Current understanding of the mechanisms of disease and therapy in multiple sclerosis has been partially derived from the model of EAE.MRI has proven to be a very sensitive technique for the detection of multiple sclerosis (MS) lesions and has provided remarkable insight into the dynamic nature of the disease process. However, our understanding of the relationship of these imaging findings to changes occurring in the tissue is rudimentary, and very few formal studies have addressed this issue. Diffusion tensor imaging (DTI) takes advantage of the anisotropic nature of water diffusion in biological samples to extract detailed micro-structural information. This technique has been widely applied for the evaluation of CNS development and pathology. A recent study indicated that directional diffusivities may serve as surrogate markers of axonal and myelin degeneration in EAE-affected mouse.The spinal cord is a common site of pathology in multiple sclerosis and is a significant contributor to clinically disability. Most MRI studies of EAE were performed on small animals such as rat or mice. The small size of the spinal cord and the presence of motion artefacts, due to cerebrospinal fluid and cardiac pulsation as well as respiration are challenging for MRI study in vivo. Up to now, only a few studies were performed on high-field magnetic resonance microimaging scanner. In view of the relatively low ratio between the RF coil and the swine cord and the recently comment that it would be beneficial to develop and characterize the EAE model in large animals. We select swine EAE model in our study. Recently high magnetic field strength (3T) clinical scanner is used. The larger core than microimaging scanner and high resolution may be a precondition to perform DTI study on spinal cord of EAE model in large animals in vivo.Objectives1. (1) Induction of Experimental Allergic Encephalomyelitis in Swine.(2) To study the spinal cord pathological changes of acute experimental allergic encephalomyelitis in swine.2. (1) To evaluate the feasibility and reproducibility of in vivo diffusion tensor imaging (DTI) of the swine spinal cord by 3T clinical scanner system.(2) To investigate the quantitative relations between DTI parameters and axonal injury pathological staining and clinical score.(3) To evaluate the sensitivity and the ability of DTI parameters to monitor axonal injury through the course of EAE.Materials and methods1. Female commercial swine, having a body weight of about 50–60 kg, were used in this study. The EAE model was induced in these swine by immunization with bovine spinal cord homogenate (SCH), complete Freund's adjuvant and M. tuberculosis.2. Neuropathological staining comprised haematoxylin and eosin (HE), luxol fast blue (LFB), and Glees silver staining. Immunohistochemical staining was performed using the Envision method. The primary antibody was polyclone anti-amyloid precursor protein (APP). LFB stains myelin, APP immunostaining demonstrate acute axonal damage and Glees silver staining detect axonal lose.3. Five control swine were imaged twice using a 3 Tesla clinical MRI unit, 6 days apart, to determine inter-assay variability. Quantitative analysis of DTI data were performed on a Syngo MRB13 imaging software platform. DTI metrics were measured in each axial section in specific ROIs placed in spinal cord at the anterior, right lateral, left lateral and posterior regions of the spinal cord at the C2/C3 vertebral level.4. Eight EAE swine (P1-P8) were used to investigate the correlation between DTI parameters and quantitative axonal injury pathological staining, Swine were performed MRI studies at distinct clinical phases and sacrificed just after the MRI study. Then spinal cords were dissected and fixed overnight in 4% paraformaldehyde for neuropathological and immunohistochemical staining. Correlations between direction diffusivity and quantitative immunohisto -chemistry staining were explored with Spearman liner correlation.5. Two EAE swine (EB, EC) were used to monitor the acute EAE onset and progression. MRI studies were set to start at 6 days after immunization. Subsequently, examinations were relatively equally spaced and in later stages adjusted to disease development. Correlations between direction diffusivity and clinical score were explored with Spearman rank correlation.Results1. Experimental allergic encephalomyelitis (EAE) was inoculated with bovin spinal cord homogenate (SCH) in the swine to create a large animal model of multiple sclerosis. The clinical course was primarily monophasic acute course with complete remission and recovery. Variables which appeared to foster disease were pertussis toxin, higher concentrations of M.tuberculosis, and intradermal inoculations.2. In our acute monophasic swine EAE model, the main histopathological changes were infiltration of inflammatory and acute axonal injury. Only a few of minor areas of demyelination and axonal lose were found at the peak point of the clinical attack. APP positive axonal and demyelination could no longer be detected by the time of clinical disease recovery completely. The numbers of axonal lose was stabile.3. All DTI parameters were compared between two measurements conducted 6 days apart. There were no statistically different at the 95% confidence level. The quantitative DTI parameters from each different region of spinal cord were not statistically different. The AD, RD, FA, and ADC were respectively equal to 1.81×10^-3 (SD = 0.06) mm²/s, 0.32×10^-3 (SD = 0.02) mm²/s, 0.72×10^-3 (SD = 0.08) and 0.95×10^-3 (SD = 0.11) mm²/s. These results were in accordance with previous studies in human spinal cord.4. The relationship between axial diffusivity and acute axonal injury was quite strong, with increasing acute axonal injury, axial diffusivity decreased (Spearman r=-0.84, P <.001). FA correlated less well with axonal injury (r=-0.31, P<0.05).5. Clear correlations between clinical disease and axial diffusivity was found in all two EAE swine respectively (r=-0.86, P<0.001 and r=-0.92, p<0.001).Conclusions1. Intradermal inoculations of bovin spinal cord homogenate was a feasibility way to inoculate acute monophasic and complete remission experimental allergic encephalomyelitis (EAE) in the swine.2. In our acute monophasic swine EAE model, the main histopathological changs were infiltration of inflammatory and acute axonal injury. Acute axonal damage may be independent of demyelination, and it's pathogensis may be different from demyelination. Our data further suggest that in an inflammatory condition like that in MS, demyelination is not a prerequisite for axonal injury.3. High quality DTI of spinal cord could be acquired by using iPAT-based echo-planar imaging technique. It is feasibility to measure DTI parameters in the axial plane of the swine spinal cord in vivo by 3T clinical scanner.4. The relationship between axial diffusivity and acute axonal injury was quite strong, with increasing acute axonal injury, axial diffusivity decreased.5. AD is a sensitive mark to monitor acute axonal injury in the course of EAE onset and progression and DTI may be a useful noninvasively tool to investigate underlying pathogenesis processes of MS and monitor the effect of experimental treatment for axonal injury.