Joanna Adamczak

High field BOLD response to forepaw stimulation in the mouse

We have established a robust protocol for longitudinal fMRI in mice at high field MRI using a medetomidine anesthesia. Electrical forepaw stimulation in anesthetized animals is widely used to produce BOLD contrast in the primary somatosensory cortex. To preserve neuronal activity, most fMRI experiments used alpha-chloralose to produce sedation, but severe side effects make this procedure unsuitable for survival experiments. As advantageous alternative, the alpha(2)-adrenergic receptor agonist medetomidine has been applied successfully to permit longitudinal fMRI studies in rats. With the advent of transgenic technology, mouse models have become increasingly attractive raising the demand for implementation of a suitable fMRI protocol for mice. Therefore, we investigated the use of medetomidine for repetitive fMRI experiments in C57BL/6 mice. We evaluated the optimal medetomidine dose for subcutaneous application. Somatosensory evoked potentials (SSEPs) in the contralateral somatosensory cortex were recorded to assess brain activity under medetominidine following forepaw stimulation. Repetitive administration of medetomidine, the requirement for longitudinal brain activation studies, was well tolerated. Using the forepaw stimulation paradigm, we observed BOLD contrast in the contralateral somatosensory cortex in approximately 50% of the performed scans using gradient echo-echo planar imaging (GE-EPI). However, imaging the small mouse brain at high field strength is challenging and we observed strong susceptibility artifacts in GE-EPI images in the cortex. We have developed an agar gel cap for successful compensation of these artifacts as prerequisite for successful mouse fMRI at 11.7T. The established protocol will be suitable for brain activation studies in transgenic animals and for studies of functional deficit and recovery after brain injury in mice.

Boosting bioluminescence neuroimaging: an optimized protocol for brain studies

Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (iv, ip, sc), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000–300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.

A multi-modality platform to image stem cell graft survival in the naïve and stroke-damaged mouse brain.

Neural stem cell implantations have been extensively investigated for treatment of brain diseases such as stroke. In order to follow the localization and functional status of cells after implantation noninvasive imaging is essential. Therefore, we developed a comprehensive multi-modality platform for in vivo imaging of graft localization, density, and survival using 19F magnetic resonance imaging in combination with bioluminescence imaging. We quantitatively analyzed cell graft survival over the first 4 weeks after transplantation in both healthy and stroke-damaged mouse brain and correlated our findings of graft vitality with the host innate immune response. The multi-modality imaging platform will help to improve cell therapy also in context other than stroke and to gain indispensable information for clinical translation.

Human neural stem cell intracerebral grafts show spontaneous early neuronal differentiation after several weeks

Human neural stem cells (hNSCs) hold great promise for the treatment of neurological diseases. Considerable progress has been made to induce neural differentiation in the cell culture in vitro and upon transplantation in vivo [2] in order to explore restoration of damaged neuronal circuits. However, in vivo conventional strategies are limited to post mortem analysis. Here, we apply our developed first fate mapping platform to monitor neuronal differentiation in vivo by magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging. Ferritin, Luciferase and GFP under neuronal-specific promoters for immature and mature neurons, respectively, were used to generate transgenic hNSCs. Differentiation-linked imaging reporter expression was validated in vitro. The time profile of spontaneous neuronal maturation after transplantation into mouse brain cortex demonstrated early neuronal differentiation within 6 weeks. Fully mature neurons expressing synaptogenesis were observed only after three months or longer. Our trimodal fate mapping strategy represents a unique non-invasive tool to monitor the time course of neuronal differentiation of transplanted stem cells in vivo.

Transient antiangiogenic treatment improves delivery of cytotoxic compounds and therapeutic outcome in lung cancer

Extensive oncologic experience argues that the most efficacious applications of antiangiogenic agents rely upon a combination with cytotoxic drugs. Yet there remains a lack of clarity about how to optimize scheduling for such drug combinations. Prudent antiangiogenic therapy might transiently normalize blood vessels to improve tumor oxygenation and drug exposure. Using [(15)O]H2O positron emission tomography imaging in a preclinical mouse model of non-small cell lung cancer, we observed that short-term treatment with the vascular endothelial growth factor receptor/platelet-derived growth factor receptor inhibitor PTK787 licensed a transient window of improved tumor blood flow. The improvement observed was associated with a reduced leakiness from tumor vessels, consistent with induction of a vascular normalization process. Initiation of a cytotoxic treatment in this window of tumor vessel normalization resulted in increased efficacy, as illustrated by improved outcomes of erlotinib administration after initial PTK787 treatment. Notably, intermittent PTK787 treatment also facilitated long-term tumor regression. In summary, our findings offer strong evidence that short-term antiangiogenic therapy can promote a transient vessel normalization process that improves the delivery and efficacy of a targeted cytotoxic drug.

A review of novel optical imaging strategies of the stroke pathology and stem cell therapy in stroke

Transplanted stem cells can induce and enhance functional recovery in experimental stroke. Invasive analysis has been extensively used to provide detailed cellular and molecular characterization of the stroke pathology and engrafted stem cells. But post mortem analysis is not appropriate to reveal the time scale of the dynamic interplay between the cell graft, the ischemic lesion and the endogenous repair mechanisms. This review describes non-invasive imaging techniques which have been developed to provide complementary in vivo information. Recent advances were made in analyzing simultaneously different aspects of the cell graft (e.g., number of cells, viability state, and cell fate), the ischemic lesion (e.g., blood–brain-barrier consistency, hypoxic, and necrotic areas) and the neuronal and vascular network. We focus on optical methods, which permit simple animal preparation, repetitive experimental conditions, relatively medium-cost instrumentation and are performed under mild anesthesia, thus nearly under physiological conditions. A selection of recent examples of optical intrinsic imaging, fluorescence imaging and bioluminescence imaging to characterize the stroke pathology and engrafted stem cells are discussed. Special attention is paid to novel optimal reporter genes/probes for genetic labeling and tracking of stem cells and appropriate transgenic animal models. Requirements, advantages and limitations of these imaging platforms are critically discussed and placed into the context of other non-invasive techniques, e.g., magnetic resonance imaging and positron emission tomography, which can be joined with optical imaging in multimodal approaches.

In vivo bioluminescence imaging of vascular remodeling after stroke.

Thrombolysis remains the only beneficial therapy for ischemic stroke, but is restricted to a short therapeutic window following the infarct. Currently research is focusing on spontaneous regenerative processes during the sub-acute and chronic phase. Angiogenesis, the formation of new blood vessels from pre-existing ones, was observed in stroke patients, correlates with longer survival and positively affects the formation of new neurons. Angiogenesis takes place in the border zones of the infarct, but further insight into the temporal profile is needed to fully apprehend its therapeutic potential and its relevance for neurogenesis and functional recovery. Angiogenesis is a multistep process, involving extracellular matrix degradation, endothelial cell proliferation, and, finally, new vessel formation. Interaction between vascular endothelial growth factor and its receptor 2 (VEGFR2) plays a central role in these angiogenic signaling cascades. In the present study we investigated non-invasively the dynamics of VEGFR2 expression following cerebral ischemia in a mouse model of middle cerebral artery occlusion (MCAO). We used a transgenic mouse expressing firefly luciferase under the control of the VEGFR2 promotor to non-invasively elucidate the temporal profile of VEGFR2 expression after stroke as a biomarker for VEGF/VEGFR2 signaling. We measured each animal repetitively up to 2 weeks after stroke and found increased VEGFR2 expression starting 3 days after the insult with peak values at 7 days. These were paralleled by increased VEGFR2 protein levels and increased vascular volume in peri-infarct areas at 14 days after the infarct, indicating that signaling via VEGFR2 leads to successful vascular remodeling. This study describes VEGFR2-related signaling is active at least up to 2 weeks after the infarct and results in increased vascular volume. Further, this study presents a novel strategy for the non-invasive evaluation of angiogenesis-based therapies.

Noninvasive Multimodal Imaging of Stem Cell Transplants in the Brain Using Bioluminescence Imaging and Magnetic Resonance Imaging

Transplantation of stem cells represents a promising approach for the therapy of different brain diseases, including stroke, Parkinsons, and Huntingtons disease. Tracking of stem cells with noninvasive imaging technologies provides insight into location, migration, and proliferation of the cells-key features for a possible clinical translation. This chapter describes a multimodal and noninvasive approach employing magnetic resonance imaging (MRI) and bioluminescence imaging (BLI), both of which offer the opportunity for repetitive measurements on the same individual, revealing the full temporal profile of cell dynamics. The combination of these modalities allows the simultaneous investigation of different aspects of the graft fate. We will present the detailed protocol for noninvasive multimodal tracking of labeled and transplanted neural stem cells, specifically optimized for brain applications, which allows repetitive assessment of localization as well as identification of cell viability and cell quantity after transplantation.

Neurogenesis upregulation on the healthy hemisphere after stroke enhances compensation for age-dependent decrease of basal neurogenesis

Stroke is a leading cause of death and disability worldwide with no treatment for the chronic phase available. Interestingly, an endogenous repair program comprising inflammation and neurogenesis is known to modulate stroke outcome. Several studies have shown that neurogenesis decreases with age but the therapeutic importance of endogenous neurogenesis for recovery from cerebral diseases has been indicated as its ablation leads to stroke aggravation and worsened outcome. A detailed characterization of the neurogenic response after stroke related to ageing would help to develop novel and targeted therapies. In an innovative approach, we used the DCX-Luc mouse, a transgenic model expressing luciferase in doublecortin-positive neuroblasts, to monitor the neurogenic response following middle cerebral artery occlusion over three weeks in three age groups (2, 6, 12months) by optical imaging while the stroke lesion was monitored by quantitative MRI. The individual longitudinal and noninvasive time profiles provided exclusive insight into age-dependent decrease in basal neurogenesis and neurogenic upregulation in response to stroke which are not accessible by conventional BrdU-based measures of cell proliferation. For cortico-striatal strokes the maximal upregulation occurred at 4days post stroke followed by a continuous decrease to basal levels by three weeks post stroke. Older animals effectively compensated for reduced basal neurogenesis by an enhanced sensitivity to the cerebral lesion, resulting in upregulated neurogenesis levels approaching those measured in young mice. In middle aged and older mice, but not in the youngest ones, additional upregulation of neurogenesis was observed in the contralateral healthy hemisphere. This further substantiates the increased propensity of older brains to respond to lesion situation. Our results clearly support the therapeutic relevance of endogenous neurogenesis for stroke recovery and particularly in older brains.

Poststroke Angiogenesis, Con Dark Side of Angiogenesis

Angiogenesis, the regrowth of new blood vessels, occurs after stroke1 and thus raises hopes that this regeneration is beneficial for patients with stroke. There is no doubt, that the restoration of blood supply, providing oxygen and nutrients to the ischemic brain tissue, is beneficial for stroke recovery. However, it is debated, whether angiogenesis truly contributes to improved recovery. Thus, fully functional new vessels occur only after several days,2 as angiogenesis is a multistep process. It is, therefore, important to have a close look at the angiogenic dynamics itself and to critically assess some pathophysiologic steps, which can be even aggravating stroke progression. Angiogenic cues arise from periods of hypoxia. Vascular endothelial growth factor (VEGF) is the most potent trigger for inducing angiogenesis and shows high upregulation as early as 1 hour after stroke. Unfortunately, it also …