Introduction

Multiple sclerosis (MS) is one of the most widespread neurological disorders with no effective therapy. The pathological features of the lesion include focal demyelination, axonal degeneration and inflammation as well as local astrocytosis and formation of astrocytic scar (Lucchinetti et al. 2005). Various animal models of MS had been developed, including those based on inducing autoimmune reaction against myelin epitopes, viral-induced encephalomyelitis and toxin-dependent focal demyelination (Rivers et al. 1933; Theiler 1934; Walczak et al. 2011). Vast majority of preclinical research on MS is based on rodent models which are very useful and cost effective. On the other hand, more clinically relevant models are needed to validate therapeutic strategies prior to clinical translation. Domestic pig (Sus scrofa domestica L.) has a brain that is gyrencephalic, only 10 times smaller compared to human (mouse is 1000 times smaller) and white matter content is similar to human brain. Convection-enhanced delivery (CED) is a technique which uses a convection with bulk flow of interstitial fluid into the brain parenchyma (Bobo et al. 1994). CED has been performed with various biochemical compounds in various preclinical models (Bankiewicz et al. 2000; Bobo et al. 1994) as well as in brain tumor patients (Ding et al. 2010). The low velocity of injection in CED minimizes potential damage caused by brain tissue displacement by the infused solution (Nduom et al. 2012) and results in uniform distribution of active ingredients during intraparenchymal injections (Kantorovich et al. 2013). Accordingly, the main goal of this study was to establish a model of focal demyelination in domestic pig brain with precise lesion placement using ClearPoint® system. Moreover, gliotoxin distribution was monitored in real-time- convection-enhanced delivery, based on gadolinium contrast.

Materials and methods

The animal procedures were performed according to ARRIVE guidelines. Eight juvenile female pigs of 25-30 kg were anesthetized with propofol (3-5mg/kg/h i.v.) and sevoflurane (0.5-1%) and following head skin incision, a burr hole was placed. The SmartFrame® and trajectory device (MRI Interventions, Inc.) was fixed to the skull using titanium screws (Fig. 1A). Gliotoxin (2-3% lysolecithin or 0.0125-0.2 mg/ml ethidium bromide in PBS) was supplemented with gadoteridol (ProHance, 2 mM). MRI compatible SmartFlow® catheter was filled with gliotoxin, injection needle was inserted beneath the dura mater and animals were placed in a 3 T MRI scanner (Magnetom Trio, Siemens). HASTE sequence was used to adjust injection trajectory to target corona radiata. After targeting, solution was infused at 250 µl/h rate over one hour with repeated T1 scans to monitor the CED. MRI follow up was performed one day and one week post injection. MRI protocol included T2 (TR/TE= 6440/83 ms) and T1 (TR/TE= 1900/2.5 ms) with and without i.v. contrast. After last imaging session, animals were transcardially perfused with 10% sucrose, followed by 4% PFA in PBS (pH= 7.4). Brains were harvested, post-fixed, cryoprotected and frozen on dry ice. Tissue specimens were used for histological analysis including eriochrome cyanine R for myelin.

Results

ClearPoint system facilitated convenient adjusting of injection trajectory under real-time MRI to navigate the needle to selected target. CED of gliotoxin/gadolinium was successfully visualized on T1w scans acquired during injection. Fig.1B shows the brain before injection and Figs. 1B1-B4 are from boxed area in B, acquired after 15, 30, 45 and 60 min show gradual expansion of the hyperintense region. MRI at one week show small region of blood-brain barrier disruption on T1+Gd scan (Fig. 1C, green arrowhead) and lesion appears as hyperintense on T2w MRI (Fig. 1D). Histological staining for myelin (Eriochrome; Fig.1E) confirmed localization of demyelination, which spatially matched with gliotoxin distribution detected on dynamic T1w and one week T2w weighted MRI (red arrowheads). Correlational analysis revealed strong correspondence between the lesion size on T2w MRI and histopathology.

Conclusion

Interventional MRI-guidance was instrumental for precise induction of focal demyelination in pig. MRI and histopathological features of lesions resemble those observed in MS.

Acknowledgements

No acknowledgement found.

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