An in vivo accelerated developmental myelination model for testing promyelinating therapeutics.

BACKGROUND: Therapeutic agents stimulating the process of myelination could be beneficial for the treatment of demyelinating diseases, such as multiple sclerosis. The efficient translation of compounds promoting myelination in vitro to efficacy in vivo is inherently time-consuming and expensive. Thyroid hormones accelerate the differentiation and maturation of oligodendrocytes, thereby promoting myelination. Systemic administration of the thyroid hormone thyroxine (T4) accelerates brain maturation, including myelination, during early postnatal development. The objective of this study was to validate an animal model for rapid testing of promyelinating therapeutic candidates for their effects on early postnatal development by using T4 as a reference compound. METHODS: Daily subcutaneous injections of T4 were given to Sprague Dawley rat pups from postnatal day (PND) 2 to PND10. Changes in white matter were determined at PND10 using diffusion tensor magnetic resonance imaging (DTI). Temporal changes in myelination from PND3 to PND11 were also assessed by quantifying myelin basic protein (MBP) expression levels in the brain using the resonance Raman spectroscopy/enzyme-linked immunosorbent assay (RRS-ELISA) and quantitative immunohistochemistry. RESULTS: DTI of white matter tracts showed significantly higher fractional anisotropy in the internal capsule of T4-treated rat pups. The distribution of total FA values in the forebrain was significantly shifted towards higher values in the T4-treated group, suggesting increased myelination. In vivo imaging data were supported by in vitro observations, as T4 administration significantly potentiated the developmental increase in MBP levels in brain lysates starting from PND8. MBP levels in the brain of animals that received treatment for 9 days correlated with the FA metric determined in the same pups in vivo a day earlier. Furthermore, accelerated developmental myelination following T4 administration was confirmed by immunohistochemical staining for MBP in coronal brain sections of treated rat pups. CONCLUSIONS: T4-treated rat pups had increased MBP expression levels and higher MRI fractional anisotropy values, both indications of accelerated myelination. This simple developmental myelination model affords a rapid test of promyelinating activity in vivo within several days, which could facilitate in vivo prescreening of candidate therapeutic compounds for developmental hypomyelinating diseases. Further research will be necessary to assess the utility of this platform for screening promyelination compounds in more complex demyelination disease models, such us multiple sclerosis.

Integrin α4 Overexpression on Rat Mesenchymal Stem Cells Enhances Transmigration and Reduces Cerebral Embolism After Intracarotid Injection.

BACKGROUND AND PURPOSE: Very late antigen-4 (integrin α4ß1)/vascular cell adhesion molecule-1 mediates leukocyte trafficking and transendothelial migration after stroke. Mesenchymal stem cells (MSCs) typically express integrin ß1 but insufficient ITGA4 (integrin α4), which limits their homing after intravascular transplantation. We tested whether ITGA4 overexpression on MSCs increases cerebral homing after intracarotid transplantation and reduces MSC-borne cerebral embolism. METHODS: Rat MSCs were lentivirally transduced to overexpress ITGA4. In vitro transendothelial migration was assessed using a Boyden chamber assay. Male Wistar rats intracarotidly received 0.5×106 control or modified MSCs 24 hours after sham or stroke surgery. In vivo behavior of MSCs in the cerebral vasculature was observed by intravital microscopy and single-photon emission computed tomography for up to 72 hours. RESULTS: Transendothelial migration of ITGA4-overexpressing MSCs was increased in vitro. MSCs were passively entrapped in microvessels in vivo and occasionally formed large cell aggregates causing local blood flow interruptions. MSCs were rarely found in perivascular niches or parenchyma at 72 hours post-transplantation, but ITGA4 overexpression significantly decreased cell aggregation and ameliorated the evoked cerebral embolism in stroke rats. CONCLUSIONS: ITGA4 overexpression on MSCs enhances transendothelial migration in vitro, but not in vivo, although it improves safety after intracarotid transplantation into stroke rats.

Lysophosphatidyl Choline Induced Demyelination in Rat Probed by Relaxation along a Fictitious Field in High Rank Rotating Frame.

In this work a new MRI modality entitled Relaxation Along a Fictitious Field in the rotating frame of rank 4 (RAFF4) was evaluated in its ability to detect lower myelin content in lysophosphatidyl choline (LPC)-induced demyelinating lesions. The lesions were induced in two areas of the rat brain with either uniform or complex fiber orientations, i.e., in the corpus callosum (cc) and dorsal tegmental tract (dtg), respectively. RAFF4 showed excellent ability to detect demyelinated lesions and good correlation with myelin content in both brain areas. In comparison, diffusion tensor imaging metrices, fractional anisotropy, mean diffusivity and axonal and radial diffusivity, and magnetization transfer (MT) metrices, longitudinal relaxation during off-resonance irradiation and MT ratio, either failed to detect demyelination in dtg or showed lower correlation with myelin density quantified from gold chloride stained histological sections. Good specifity of RAFF4 to myelin was confirmed by its low correlation with cell density assesed from Nissl stained sections as well as its lack of sensitivity to pH changes in the physiological range as tested in heat denaturated bovine serum albumin phantoms. The excellent ability of RAFF4 to detect myelin content and its insensitivity to fiber orientation distribution, gliosis and pH, together with low specific absorption rate, demonstrates the promise of rotating frame of rank n (RAFFn) as a valuable MRI technique for non-invasive imaging of demyelinating lesions.