Mathias Hoehn

Monitoring of implanted stem cell migration in vivo: A highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat

In vivo monitoring of stem cells after grafting is essential for a better understanding of their migrational dynamics and differentiation processes and of their regeneration potential. Migration of endogenous or grafted stem cells and neurons has been described in vertebrate brain, both under normal conditions from the subventricular zone along the rostral migratory stream and under pathophysiological conditions, such as degeneration or focal cerebral ischemia. Those studies, however, relied on invasive analysis of brain sections in combination with appropriate staining techniques. Here, we demonstrate the observation of cell migration under in vivo conditions, allowing the monitoring of the cell dynamics within individual animals, and for a prolonged time. Embryonic stem (ES) cells, constitutively expressing the GFP, were labeled by a lipofection procedure with a MRI contrast agent and implanted into rat brains. Focal cerebral ischemia had been induced 2 weeks before implantation of ES cells into the healthy, contralateral hemisphere. MRI at 78-μm isotropic spatial resolution permitted the observation of the implanted cells with high contrast against the host tissue, and was confirmed by GFP registration. During 3 weeks, cells migrated along the corpus callosum to the ventricular walls, and massively populated the borderzone of the damaged brain tissue on the hemisphere opposite to the implantation sites. Our results indicate that ES cells have high migrational dynamics, targeted to the cerebral lesion area. The imaging approach is ideally suited for the noninvasive observation of cell migration, engraftment, and morphological differentiation at high spatial and temporal resolution.

Host-Dependent Tumorigenesis of Embryonic Stem Cell Transplantation in Experimental Stroke

The therapeutical potential of transplantation of undifferentiated and predifferentiated murine embryonic stem cells for the regeneration of the injured brain was investigated in two rodent stroke models. Undifferentiated embryonic stem cells xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury migrated along the corpus callosum towards the damaged tissue and differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. The authors demonstrated a hitherto unrecognized inverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells, which raises concerns about safety provisions when the therapeutical potential of human embryonic stem cells is tested in preclinical animal models.

Cellular MR Imaging

Cellular MR imaging is a young field that aims to visualize targeted cells in living organisms. In order to provide a different signal intensity of the targeted cell, they are either labeled with MR contrast agents in vivo or prelabeled in vitro. Either (ultrasmall) superparamagnetic iron oxide [(U)SPIO] particles or (polymeric) paramagnetic chelates can be used for this purpose. For in vivo cellular labeling, Gd3+- and Mn2+- chelates have mainly been used for targeted hepatobiliary imaging, and (U)SPIO-based cellular imaging has been focused on imaging of macrophage activity. Several of these magneto-pharmaceuticals have been FDA-approved or are in late-phase clinical trials. As for prelabeling of cells in vitro, a challenge has been to induce a sufficient uptake of contrast agents into nonphagocytic cells, without affecting normal cellular function. It appears that this issue has now largely been resolved, leading to an active research on monitoring the cellular biodistribution in vivo following transplantation or transfusion of these cells, including cell migration and trafficking. New applications of cellular MR imaging will be directed, for instance, towards our understanding of hematopoietic (immune) cell trafficking and of novel guided (stem) cell-based therapies aimed to be translated to the clinic in the future.

Locus Ceruleus Degeneration Promotes Alzheimer Pathogenesis in Amyloid Precursor Protein 23 Transgenic Mice

Locus ceruleus (LC) degeneration and loss of cortical noradrenergic innervation occur early in Alzheimer’s disease (AD). Although this has been known for several decades, the contribution of LC degeneration to AD pathogenesis remains unclear. We induced LC degeneration with N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (dsp4) in amyloid precursor protein 23 (APP23) transgenic mice with a low amyloid load. Then 6 months later the LC projection areas showed a robust elevation of glial inflammation along with augmented amyloid plaque deposits. Moreover, neurodegeneration and neuronal loss significantly increased. Importantly, the paraventricular thalamus, a nonprojection area, remained unaffected. Radial arm maze and social partner recognition tests revealed increased memory deficits while high-resolution magnetic resonance imaging-guided micro-positron emission tomography demonstrated reduced cerebral glucose metabolism, disturbed neuronal integrity, and attenuated acetylcholinesterase activity. Nontransgenic mice with LC degeneration were devoid of these alterations. Our data demonstrate that the degeneration of LC affects morphology, metabolism, and function of amyloid plaque-containing higher brain regions in APP23 mice. We postulate that LC degeneration substantially contributes to AD development.

A fully noninvasive and robust experimental protocol for longitudinal fMRI studies in the rat

Functional magnetic resonance imaging (fMRI) is a unique tool to study brain activity and plasticity changes. Combination of blood-oxygen level-dependent (BOLD) fMRI and electrical forepaw stimulation has been used as a standard model to study the somatosensory pathway and brain rehabilitation in rats. The majority of fMRI studies have been performed in animals anesthetized with alpha-chloralose as functional-metabolic coupling is best preserved under this anesthesia. However, alpha-chloralose is not suitable for survival procedures due to side effects, limiting its use to single time point studies of the same animal. We therefore developed a new, totally noninvasive fMRI protocol, using sedation with the alpha2-adrenoreceptor agonist medetomidine in combination with transcutaneous monitoring of blood gases. The continuous subcutaneous administration of medetomidine resulted in stable physiological conditions over a long time and all animals tolerated the repetitive fMRI experiments well. A robust and reproducible, significant BOLD signal increase was observed upon forepaw stimulation in the contralateral primary somatosensory cortex in two consecutive medetomidine sessions in all rats, which was similar to the BOLD signal increase observed in the same animals under alpha-chloralose during a third independent session. Activation in the secondary somatosensory cortex was observed less frequently under both medetomidine and alpha-chloralose. No head motion artifacts or nonspecific brain activation was present. Sedation was quickly reversed by the administration of the antagonist atipamezole after the fMRI experiment. These results demonstrate that longitudinal fMRI studies can be performed safely under sedation with medetomidine to study functional recovery processes upon therapeutical treatment.

Application of magnetic resonance to animal models of cerebral ischemia

The present review has been compiled to highlight the role of magnetic resonance imaging (MRI) and MR spectroscopy (MRS) for the investigation of cerebral ischemia in the animal experimental field of basic research. We have focused on stroke investigations analyzing the pathomechanisms of the disease evolution and on new advances in both nuclear MR (NMR) methodology or genetic engineering of transgenic animals for the study of complex molecular relationships and causes of the disease. Furthermore, we have tried to include metabolic and genetic aspects, as well as the application of functional imaging, for the investigation of the disturbance or restitution of functional brain activation under pathological conditions as relates to controlled animal experiments. J. Magn. Reson. Imaging 2001;14:491–509.

Early Prediction of Functional Recovery after Experimental Stroke: Functional Magnetic Resonance Imaging, Electrophysiology, and Behavioral Testing in Rats

Therapeutic success of treatment of cerebral diseases must be assessed in terms of functional outcome. In experimental stroke studies, this has been limited to behavioral studies combined with morphological evaluations and single time point functional magnetic resonance imaging (fMRI) measurements but lacking the access to understanding underlying mechanisms for alterations in brain activation. Using a recently developed blood oxygenation level-dependent fMRI protocol to study longitudinal and intraindividual profiles of functional brain activation in the somatosensory system, we have demonstrated activation reemergence in the original representation field as the basic principle of functional recovery from experimental stroke. No plastic reorganization has been observed at any time point during 7 weeks after stroke induction. Applying combined recording of fMRI and somatosensory evoked potentials, we observed a tight coupling of electrical brain activity and hemodynamic response at all times, indicating persistent preservation of neurovascular coupling. Identification of functional brain recovery mechanisms has important implications for the understanding of brain plasticity after cerebral lesions, whereas preservation of neurovascular coupling is important for the clinical translation of fMRI.

Histochemical detection of ultrasmall superparamagnetic iron oxide (USPIO) contrast medium uptake in experimental brain ischemia

Recently, macrophage infiltration in different central nervous system (CNS) pathologies has been visualized with ultrasmall particles of iron oxide (USPIO) as a new cell‐specific contrast medium for MRI. However, validation of these findings at the histological level has been hampered by the fact that the in situ detection of iron uptake by conventional Prussian blue staining is not sensitive enough to detect low amounts of iron in the brain. Here, an improved method for the histochemical detection of USPIO uptake in ischemic brain lesions is reported. The procedure relies on the sequential enhancement of Prussian blue staining by diaminobenzidine and silver/gold impregnation. After photothrombotic cortical brain infarction, this method allowed sensitive in situ detection of iron‐laden macrophages which matched both macrophage immunostaining and USPIO‐induced signal alterations in high‐resolution 7 T MRI. This staining method provides a basis for correlative histological assessment of USPIO‐enhanced MRI in a broad spectrum of CNS pathologies. Magn Reson Med 52:403–406, 2004.

Noninvasive Imaging of Endogenous Neural Stem Cell Mobilization In Vivo Using Positron Emission Tomography

Neural stem cells reside in two major niches in the adult brain [i.e., the subventricular zone (SVZ) and the dentate gyrus of the hippocampus]. Insults to the brain such as cerebral ischemia result in a physiological mobilization of endogenous neural stem cells. Since recent studies showed that pharmacological stimulation can be used to expand the endogenous neural stem cell niche, hope has been raised to enhance the brains own regenerative capacity. For the evaluation of such novel therapeutic approaches, longitudinal and intraindividual monitoring of the endogenous neural stem cell niche would be required. However, to date no conclusive imaging technique has been established. We used positron emission tomography (PET) and the radiotracer 3′-deoxy-3′-[18F]fluoro-l-thymidine ([18F]FLT) that enables imaging and measuring of proliferation to noninvasively detect endogenous neural stem cells in the normal and diseased adult rat brain in vivo. This method indeed visualized neural stem cell niches in the living rat brain, identified as increased [18F]FLT-binding in the SVZ and the hippocampus. Focal cerebral ischemia and subsequent damage of the blood–brain barrier did not interfere with the capability of [18F]FLT-PET to visualize neural stem cell mobilization. Moreover, [18F]FLT-PET allowed for an in vivo quantification of increased neural stem cell mobilization caused by pharmacological stimulation or by focal cerebral ischemia. The data suggest that noninvasive longitudinal monitoring and quantification of endogenous neural stem cell activation in the brain is feasible and that [18F]FLT-PET could be used to monitor the effects of drugs aimed at expanding the neural stem cell niche.

Labeling cells for in vivo tracking using 19F MRI

Noninvasive in vivo cell tracking is crucial to fully understand the function of mobile and/or transplanted cells, particularly immune cells and cellular therapeutics. (19)F MRI for cell tracking has several advantages; chief among them are its noninvasive nature which allows longitudinal data acquisition, use of a stable, non-radioactive isotope permitting long-term tracking, the absence of confounding endogenous signal, and the ability to quantify cell numbers from image data. However, generation of sufficient signal i.e. (19)F cell loading is a key challenge, particularly with non-phagocytic cells such as lymphocytes and stem cells. A range of (19)F cell labels have been developed, including emulsions, particles, polymers, and agents for clinical use. Various animal and primary human cells, such as dendritic cells, lymphocytes and phagocytes have been successfully labeled and studied in models of autoimmune disease, inflammation and transplant rejection. Primary human cells, particularly dendritic cells as used in vaccine therapy have been tested for imminent clinical application. Here, we summarize current cell loading strategies and sensitivity of in vivo cell imaging with (19)F MRI, and discuss the processing of image data for accurate quantification of cell numbers. This novel technology is uniquely applicable to the longitudinal and quantitative tracking of cells in vivo.