Changes in interstitial fluid flow, mass transport and the bone cell response in microgravity and normogravity.

In recent years, our scientific interest in spaceflight has grown exponentially and resulted in a thriving area of research, with hundreds of astronauts spending months of their time in space. A recent shift toward pursuing territories farther afield, aiming at near-Earth asteroids, the Moon, and Mars combined with the anticipated availability of commercial flights to space in the near future, warrants continued understanding of the human physiological processes and response mechanisms when in this extreme environment. Acute skeletal loss, more severe than any bone loss seen on Earth, has significant implications for deep space exploration, and it remains elusive as to why there is such a magnitude of difference between bone loss on Earth and loss in microgravity. The removal of gravity eliminates a critical primary mechano-stimulus, and when combined with exposure to both galactic and solar cosmic radiation, healthy human tissue function can be negatively affected. An additional effect found in microgravity, and one with limited insight, involves changes in dynamic fluid flow. Fluids provide the most fundamental way to transport chemical and biochemical elements within our bodies and apply an essential mechano-stimulus to cells. Furthermore, the cell cytoplasm is not a simple liquid, and fluid transport phenomena together with viscoelastic deformation of the cytoskeleton play key roles in cell function. In microgravity, flow behavior changes drastically, and the impact on cells within the porous system of bone and the influence of an expanding level of adiposity are not well understood. This review explores the role of interstitial fluid motion and solute transport in porous bone under two different conditions: normogravity and microgravity.

High-Density Electrical Recording and Impedance Imaging With a Multi-Modal CMOS Multi-Electrode Array Chip.

Multi-electrode arrays, both active or passive, emerged as ideal technologies to unveil intricated electrophysiological dynamics of cells and tissues. Active MEAs, designed using complementary metal oxide semiconductor technology (CMOS), stand over passive devices thanks to the possibility of achieving single-cell resolution, the reduced electrode size, the reduced crosstalk and the higher functionality and portability. Nevertheless, most of the reported CMOS MEA systems mainly rely on a single operational modality, which strongly hampers the applicability range of a single device. This can be a limiting factor considering that most biological and electrophysiological dynamics are often based on the synergy of multiple and complex mechanisms acting from different angles on the same phenomena. Here, we designed a CMOS MEA chip with 16,384 titanium nitride electrodes, 6 independent operational modalities and 1,024 parallel recording channels for neuro-electrophysiological studies. Sixteen independent active areas are patterned on the chip surface forming a 4 × 4 matrix, each one including 1,024 electrodes. Electrodes of four different sizes are present on the chip surface, ranging from 2.5 × 3.5 µm2 up to 11 × 11.0 µm2, with 15 µm pitch. In this paper, we exploited the impedance monitoring and voltage recording modalities not only to monitor the growth and development of primary rat hippocampal neurons, but also to assess their electrophysiological activity over time showing a mean spike amplitude of 144.8 ± 84.6 µV. Fixed frequency (1 kHz) and high sampling rate (30 kHz) impedance measurements were used to evaluate the cellular adhesion and growth on the chip surface. Thanks to the high-density configuration of the electrodes, as well as their dimension and pitch, the chip can appreciate the evolutions of the cell culture morphology starting from the moment of the seeding up to mature culture conditions. The measurements were confirmed by fluorescent staining. The effect of the different electrode sizes on the spike amplitudes and noise were also discussed. The multi-modality of the presented CMOS MEA allows for the simultaneous assessment of different physiological properties of the cultured neurons. Therefore, it can pave the way both to answer complex fundamental neuroscience questions as well as to aid the current drug-development paradigm.

Pixel super-resolution for lens-free holographic microscopy using deep learning neural networks.

Lens-free holographic microscopy (LFHM) provides a cost-effective tool for large field-of-view imaging in various biomedical applications. However, due to the unit optical magnification, its spatial resolution is limited by the pixel size of the imager. Pixel super-resolution (PSR) technique tackles this problem by using a series of sub-pixel shifted low-resolution (LR) lens-free holograms to form the high-resolution (HR) hologram. Conventional iterative PSR methods require a large number of measurements and a time-consuming reconstruction process, limiting the throughput of LFHM in practice. Here we report a deep learning-based PSR approach to enhance the resolution of LFHM. Compared with the existing PSR methods, our neural network-based approach outputs the HR hologram in an end-to-end fashion and maintains consistency in resolution improvement with a reduced number of LR holograms. Moreover, by exploiting the resolution degradation model in the imaging process, the network can be trained with a data set synthesized from the LR hologram itself without resorting to the HR ground truth. We validated the effectiveness and the robustness of our method by imaging various types of samples using a single network trained on an entirely different data set. This deep learning-based PSR approach can significantly accelerate both the data acquisition and the HR hologram reconstruction processes, therefore providing a practical solution to fast, lens-free, super-resolution imaging.

Burmese python target reflectivity compared to natural Florida foliage background reflectivity.

The Florida Everglades is infested with Burmese pythons caused by the release of exotic pets in the 1980s. The current estimates are between 30,000 and 300,000 pythons, where the result is a severe decline in Everglade mammals: 90% reductions in raccoon, opossum, bobcats, and foxes. The marsh rabbits are completely gone. The population of the pythons is rapidly increasing exponentially with 20-50 eggs per snake with a life span of up to 20 years. Pythons have been captured in the Everglades with lengths of nearly 6 m. Researchers in the state of Florida are concerned that these pythons are (1) permanently damaging the Everglades, (2) migrating further north into populated areas of Florida, and (3) endangering wildlife, pets, and eventually, people. There have been a number of sensing efforts attempted in the large-area detection of pythons, where limited success has been achieved. For example, infrared sensors have been applied to the problem, but the pythons are cold-blooded, so the infrared bands do not work well. Imec has leveraged its expertise and infrastructure in semiconductor processing to produce highly compact, higher performance, and relatively cheaper hyperspectral image sensors and camera systems. In this work, Imec teamed with the University of Florida and Extended Reality Systems to obtain hyperspectral reflectivity measurements of Burmese pythons along with natural Florida background foliage to determine bands or band combinations that may be exploited in the large-area detection of pythons. The bands investigated are the visible-near infrared (or VisNIR) and the shortwave infrared (SWIR) bands. The results show that there are enough differences in the data collection such that a single band, inexpensive VisNIR band camera may provide reasonable results and a two-band, VisNIR/SWIR combination may provide higher performance results. In this paper, we provide the VisNIR results.

Reflective lens-free imaging on high-density silicon microelectrode arrays for monitoring and evaluation of in vitro cardiac contractility.

The high rate of drug attrition caused by cardiotoxicity is a major challenge for drug development. Here, we developed a reflective lens-free imaging (RLFI) approach to non-invasively record in vitro cell deformation in cardiac monolayers with high temporal (169 fps) and non-reconstructed spatial resolution (352 µm) over a field-of-view of maximally 57 mm2. The method is compatible with opaque surfaces and silicon-based devices. Further, we demonstrated that the system can detect the impairment of both contractility and fast excitation waves in cardiac monolayers. Additionally, the RLFI device was implemented on a CMOS-based microelectrode array to retrieve multi-parametric information of cardiac cells, thereby offering more in-depth analysis of drug-induced (cardiomyopathic) effects for preclinical cardiotoxicity screening applications.

Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.

Time-lapse imaging of biological samples is important for understanding complex (patho)physiological processes. A growing number of point-of-care biomedical assays rely on real-time imaging of flowing or migrating cells. However, the cost and complexity of integrating experimental models simulating physiologically relevant microenvironments with bulky imaging systems that offer sufficient spatiotemporal resolution limit the use of time-lapse assays in research and clinical settings. This paper introduces a compact and affordable lens-free imaging (LFI) device based on the principle of coherent in-line, digital holography for time-lapse cell migration assays. The LFI device combines single-cell resolution (1.2 µm) with a large field of view (6.4 × 4.6 mm(2)), thus rendering it ideal for high-throughput applications and removing the need for expensive and bulky programmable motorized stages. The set-up is so compact that it can be housed in a standard cell culture incubator, thereby avoiding custom-built stage top incubators. LFI is thoroughly benchmarked against conventional live-cell phase contrast microscopy for random cell motility on two-dimensional (2D) surfaces and confined migration on 1D-microprinted lines and in microchannels using breast adenocarcinoma cells. The quality of the results obtained by the two imaging systems is comparable, and they reveal that cells migrate more efficiently upon increasing confinement. Interestingly, assays of confined migration more readily distinguish the migratory potential of metastatic MDA-MB-231 cells from non-metastatic MCF7 cells relative to traditional 2D migration assays. Altogether, this single-cell migration study establishes LFI as an elegant and useful tool for live-cell imaging.

Long-Term Fate Mapping Using Conditional Lentiviral Vectors Reveals a Continuous Contribution of Radial Glia-Like Cells to Adult Hippocampal Neurogenesis in Mice.

Newborn neurons are generated throughout life in two neurogenic regions, the subventricular zone and the hippocampal dentate gyrus. Stimulation of adult neurogenesis is considered as an attractive endogenous repair mechanism to treat different neurological disorders. Although tremendous progress has been made in our understanding of adult hippocampal neurogenesis, important questions remain unanswered, regarding the identity and the behavior of neural stem cells in the dentate gyrus. We previously showed that conditional Cre-Flex lentiviral vectors can be used to label neural stem cells in the subventricular zone and to track the migration of their progeny with non-invasive bioluminescence imaging. Here, we applied these Cre-Flex lentiviral vectors to study neurogenesis in the dentate gyrus with bioluminescence imaging and histological techniques. Stereotactic injection of the Cre-Flex vectors into the dentate gyrus of transgenic Nestin-Cre mice resulted in specific labeling of the nestin-positive neural stem cells. The labeled cell population could be detected with bioluminescence imaging until 9 months post injection, but no significant increase in the number of labeled cells over time was observed with this imaging technique. Nevertheless, the specific labeling of the nestin-positive neural stem cells, combined with histological analysis at different time points, allowed detailed analysis of their neurogenic potential. This long-term fate mapping revealed that a stable pool of labeled nestin-positive neural stem cells continuously contributes to the generation of newborn neurons in the mouse brain until 9 months post injection. In conclusion, the Cre-Flex technology is a valuable tool to address remaining questions regarding neural stem cell identity and behavior in the dentate gyrus.

Bioluminescence imaging of stroke-induced endogenous neural stem cell response.

Brain injury following stroke affects neurogenesis in the adult mammalian brain. However, a complete understanding of the origin and fate of the endogenous neural stem cells (eNSCs) in vivo is missing. Tools and technology that allow non-invasive imaging and tracking of eNSCs in living animals will help to overcome this hurdle. In this study, we aimed to monitor eNSCs in a photothrombotic (PT) stroke model using in vivo bioluminescence imaging (BLI). In a first strategy, inducible transgenic mice expressing firefly luciferase (Fluc) in the eNSCs were generated. In animals that received stroke, an increased BLI signal originating from the infarct region was observed. However, due to histological limitations, the identity and exact origin of cells contributing to the increased BLI signal could not be revealed. To overcome this limitation, we developed an alternative strategy employing stereotactic injection of conditional lentiviral vectors (Cre-Flex LVs) encoding Fluc and eGFP in the subventricular zone (SVZ) of Nestin-Cre transgenic mice, thereby specifically labeling the eNSCs. Upon induction of stroke, increased eNSC proliferation resulted in a significant increase in BLI signal between 2days and 2weeks after stroke, decreasing after 3months. Additionally, the BLI signal relocalized from the SVZ towards the infarct region during the 2weeks following stroke. Histological analysis at 90days post stroke showed that in the peri-infarct area, 36% of labeled eNSC progeny differentiated into astrocytes, while 21% differentiated into mature neurons. In conclusion, we developed and validated a novel imaging technique that unequivocally demonstrates that nestin(+) eNSCs originating from the SVZ respond to stroke injury by increased proliferation, migration towards the infarct region and differentiation into both astrocytes and neurons. In addition, this new approach allows non-invasive and specific monitoring of eNSCs over time, opening perspectives for preclinical evaluation of candidate stroke therapeutics.

Current challenges for the advancement of neural stem cell biology and transplantation research.

Transplantation of neural stem cells (NSC) is hoped to become a promising primary or secondary therapy for the treatment of various neurodegenerative disorders of the central nervous system (CNS), as demonstrated by multiple pre-clinical animal studies in which functional recovery has already been demonstrated. However, for NSC therapy to be successful, the first challenge will be to define a transplantable cell population. In the first part of this review, we will briefly discuss the main features of ex vivo culture and characterisation of NSC. Next, NSC grafting itself may not only result in the regeneration of lost tissue, but more importantly has the potential to improve functional outcome through many bystander mechanisms. In the second part of this review, we will briefly discuss several pre-clinical studies that contributed to a better understanding of the therapeutic potential of NSC grafts in vivo. However, while many pre-clinical animal studies mainly report on the clinical benefit of NSC grafting, little is known about the actual in vivo fate of grafted NSC. Therefore, the third part of this review will focus on non-invasive imaging techniques for monitoring cellular grafts in the brain under in vivo conditions. Finally, as NSC transplantation research has evolved during the past decade, it has become clear that the host micro-environment itself, either in healthy or injured condition, is an important player in defining success of NSC grafting. The final part of this review will focus on the host environmental influence on survival, migration and differentiation of grafted NSC.

Depletion of PINK1 affects mitochondrial metabolism, calcium homeostasis and energy maintenance.

Loss-of-function mutations in the gene encoding the mitochondrial PTEN-induced putative kinase 1 (PINK1) are a major cause of early-onset familial Parkinson's disease (PD). Recent studies have highlighted an important function for PINK1 in clearing depolarized mitochondria by mitophagy. However, the role of PINK1 in mitochondrial and cellular functioning in physiological conditions is still incompletely understood. Here, we investigate mitochondrial and cellular calcium (Ca(2+)) homeostasis in PINK1-knockdown and PINK1-knockout mouse cells, both in basal metabolic conditions and after physiological stimulation, using unbiased automated live single-cell imaging in combination with organelle-specific fluorescent probes. Our data reveal that depletion of PINK1 induces moderate fragmentation of the mitochondrial network, mitochondrial membrane depolarization and increased production of reactive oxygen species. This results in reduced uptake of Ca(2+) by mitochondria after physiological stimulation. As a consequence, cells with knockdown or knockout of PINK1 display impaired mitochondrial ATP synthesis, which is exacerbated under conditions of increased ATP demand, thereby affecting cytosolic Ca(2+) extrusion. The impairment in energy maintenance was confirmed in the brain of PINK1-knockout mice by in vivo bioluminescence imaging. Our findings demonstrate a key role for PINK1 in the regulation of mitochondrial homeostasis and energy metabolism under physiological conditions.

Efficient gene transfer into the mouse lung by fetal intratracheal injection of rAAV2/6.2.

Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for ß-galactosidase (ß-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

Immunohistochemical detection of transgene expression in the brain using small epitope tags.

BACKGROUND: In vivo overexpression of proteins is a powerful approach to study their biological function, generate disease models or evaluate gene therapy approaches. In order to investigate an exogenously expressed protein, specific and sensitive detection is essential. Unfortunately, antibodies that allow histological detection of the protein of interest are not always readily available. The use of an epitope tag fused to the protein can circumvent this problem as well as provide the possibility to discriminate endogenous from overexpressed proteins. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags. RESULTS: In the present study, we evaluated several small epitope tags together with corresponding anti-tag antibodies for the detection of overexpressed proteins in rat brain, using eGFP as a reference. We generated several lentiviral vectors encoding eGFP with different N-terminally fused small epitope tags (AU1, flag, 3flag, HA, myc and V5). After confirmation of their functionality in cell culture, we injected these lentiviral vectors stereotactically into the striatum of rats and prepared paraformaldehyde fixed floating sections for immunohistochemical analysis. Using multiple antibodies and antibody dilutions per epitope tag, we extensively assessed the efficiency of several anti-tag antibodies for chromogenic immunohistochemical detection of the epitope tagged eGFPs by determining the proportion of immunoreactivity detected by anti-tag antibodies compared to anti-GFP antibody. Using fluorescence immunohistochemistry and confocal microscopy, we also quantified the proportion of eGFP-positive cells detected by anti-tag antibodies. Our results show that all the examined small epitope tags could be detected by anti-tag antibodies both in cell extracts as well as in vivo, although to varying degrees depending on the tag and antibody used. Using the presented protocol, V5/anti-V5 and HA/HA11 tag/antibody combinations provided the most sensitive detection in brain tissue. We confirmed the applicability of these optimized in vivo tag detection conditions for a difficult to detect protein, firefly luciferase (fLuc), using lentiviral vector constructs expressing V5 tagged and 3flag tagged fLuc protein. CONCLUSIONS: We show here that several small epitope tags are useful for immunohistochemical detection of exogenous proteins in vivo. Our study also provides a generic methodology which is broadly applicable for the detection of overexpressed transgenes in mammalian brain tissue.

Inhibition of FK506 binding proteins reduces alpha-synuclein aggregation and Parkinson's disease-like pathology.

alpha-Synuclein (alpha-SYN) is a key player in the pathogenesis of Parkinson's disease (PD). In pathological conditions, the protein is present in a fibrillar, aggregated form inside cytoplasmic inclusions called Lewy bodies. Members of the FK506 binding protein (FKBP) family are peptidyl-prolyl isomerases that were shown recently to accelerate the aggregation of alpha-SYN in vitro. We now established a neuronal cell culture model for synucleinopathy based on oxidative stress-induced alpha-SYN aggregation and apoptosis. Using high-content analysis, we examined the role of FKBPs in aggregation and apoptotic cell death. FK506, a specific inhibitor of this family of proteins, inhibited alpha-SYN aggregation and neuronal cell death in this synucleinopathy model dose dependently. Knockdown of FKBP12 or FKBP52 reduced the number of alpha-SYN aggregates and protected against cell death, whereas overexpression of FKBP12 or FKBP52 accelerated both aggregation of alpha-SYN and cell death. Thus, FK506 likely targets FKBP members in the cell culture model. Furthermore, oral administration of FK506 after viral vector-mediated overexpression of alpha-SYN in adult mouse brain significantly reduced alpha-SYN aggregate formation and neuronal cell death. Our data explain previously described neuroregenerative and neuroprotective effects of immunophilin ligands and validate FKBPs as a novel drug target for the causative treatment of PD.

Impaired neurogenesis, learning and memory and low seizure threshold associated with loss of neural precursor cell survivin.

BACKGROUND: Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family in that it exhibits antiapoptotic properties and also promotes the cell cycle and mediates mitosis as a chromosome passenger protein. Survivin is highly expressed in neural precursor cells in the brain, yet its function there has not been elucidated. RESULTS: To examine the role of neural precursor cell survivin, we first showed that survivin is normally expressed in periventricular neurogenic regions in the embryo, becoming restricted postnatally to proliferating and migrating NPCs in the key neurogenic sites, the subventricular zone (SVZ) and the subgranular zone (SGZ). We then used a conditional gene inactivation strategy to delete the survivin gene prenatally in those neurogenic regions. Lack of embryonic NPC survivin results in viable, fertile mice (SurvivinCamcre) with reduced numbers of SVZ NPCs, absent rostral migratory stream, and olfactory bulb hypoplasia. The phenotype can be partially rescued, as intracerebroventricular gene delivery of survivin during embryonic development increases olfactory bulb neurogenesis, detected postnatally. SurvivinCamcre brains have fewer cortical inhibitory interneurons, contributing to enhanced sensitivity to seizures, and profound deficits in memory and learning. CONCLUSIONS: The findings highlight the critical role that survivin plays during neural development, deficiencies of which dramatically impact on postnatal neural function.

Seeing genes at work in the living brain with non-invasive molecular imaging.

Over the past ten years, a variety of imaging techniques have been developed that allow non-invasive detection of gene expression within the brain of intact mammals, ranging from mouse to man. The basic concepts of these imaging techniques, including positron emission tomography, single photon emission computed tomography, magnetic resonance imaging and spectroscopy, bioluminescence imaging and fluorescent imaging, are discussed. The expression of imaging reporter genes can be detected and quantified by these imaging techniques, which allow to unravel the temporospatial dynamics of gene expression within the intact living animal. Different imaging reporter genes have been developed each with their specific use in the basic and clinical neurosciences. Applications of reporter gene imaging can be found in neurooncology, infectious disease of the central nervous system, brain gene transfer, neural cellular therapy and in transgenic mice. Strategies that aim to image gene expression based on detection of mRNA levels have also been developed. We anticipate that these techniques will have a strong impact on preclinical neuroscience and will be of utmost importance in the implementation of gene and cell therapy for diseases of the brain.

Highly efficient multicistronic lentiviral vectors with peptide 2A sequences.

Gene discovery and gene therapy call for advanced technologies to reliably assess gene expression; efficient coupling of gene expression to the expression of reporter genes is critical. Various noninvasive molecular imaging modalities have emerged to track biological processes in animal models. Here, we evaluate various strategies to link transgene expression with that of an (imaging) reporter gene. Using lentiviral vectors containing internal ribosomal entry sites (IRES), 2A-like peptides, or a bidirectional promoter, we compared their ability to ensure efficient coexpression of multiple reporter genes. Although the encephalomyocarditis virus (EMCV) IRES yielded functional bicistronic vectors, the expression level of the reporter downstream of IRES was consistently lower than that of the upstream transgene. Interestingly, peptide 2A constructs performed best in vitro and in vivo, providing effective noninvasive follow-up of transgene expression and having reporter gene expression levels in line with that of the single reporter constructs. The intrinsic "cleavage" property of the peptide 2A sequences allows each protein to be produced at proportional levels, opening ample possibilities for functional genomics and future gene therapeutic applications. Last, using various peptide 2A sequences, we engineered the triple reporter LV-3R (i.e., eGFP, fLuc, HSV1-sr39tk), enabling efficient multimodality readouts in vivo.

Regulation of astrocyte inflammatory responses by the Parkinson's disease-associated gene DJ-1.

The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.

Noninvasive and quantitative monitoring of adult neuronal stem cell migration in mouse brain using bioluminescence imaging.

It is now generally accepted that continuous neurogenesis occurs in the adult mammalian brain, including that of humans. Modulation of adult neurogenesis can provide therapeutic benefits for various brain disorders, including stroke and Parkinson's disease. The subventricular zone-olfactory bulb pathway is one of the preferred model systems by which to study neural stem cell proliferation, migration, and differentiation in adult rodent brain. Research on adult neurogenesis would greatly benefit from reliable methods for long-term noninvasive in vivo monitoring. We have used lentiviral vectors encoding firefly luciferase to stably mark endogenous neural stem cells in the mouse subventricular zone. We show that bioluminescence imaging (BLI) allows quantitative follow-up of the migration of adult neural stem cells into the olfactory bulb in time. Moreover, we propose a model to fit the kinetic data that allows estimation of migration and survival times of the neural stem cells using in vivo BLI. Long-term expression of brain-derived neurotrophic factor in the subventricular zone attenuated neurogenesis, as detected by histology and BLI. In vivo monitoring of the impact of drugs or genes on adult neurogenesis is now within reach.

Fetal gene transfer with lentiviral vectors: long-term in vivo follow-up evaluation in a rat model.

OBJECTIVE: The purpose of this study was to evaluate the long-term expression of a transgene and subsequent immune response after the injection of lentiviral vectors in a fetal rats. STUDY DESIGN: Fetal rats were injected in the liver, peritoneal cavity, or lung at E19 (term, E21) with a lentiviral vector expressing enhanced green fluorescent protein and luciferase. Controls received saline solution. After birth, full body bioluminescence was done at weeks 1, 4, 10, and 30 of life; seroconversion for the transgene was assessed. RESULTS: All surviving fetuses that had been injected in the liver (8/9 fetuses), peritoneum (3/3 fetuses), or lung (9/10 fetuses) showed a signal on bioluminescence imaging scan up to 30 weeks. None of the survivors displayed seroconversion against the transgene. CONCLUSION: In the rat model, the administration of lentiviral vectors into the fetal lung and liver resulted in long-term transgene expression without detectable humoral immune response.

Noninvasive monitoring of long-term lentiviral vector-mediated gene expression in rodent brain with bioluminescence imaging.

Gene transfer into the central nervous system is an emerging therapeutic strategy for a range of neurological diseases, including neurodegeneration. This approach would benefit from imaging technologies that could determine the extent, magnitude, and duration of transgene expression. We have used bioluminescence imaging (BLI) to image lentiviral vector-mediated gene transfer into the mouse brain. We constructed human immunodeficiency virus type 1 lentiviral vectors that encode firefly luciferase and transduce cells in culture. After stereotactic injection of these vectors into the brain, we were able to detect luciferase expression in living mice and rats. We characterized the signal in mouse brain in terms of localization, kinetics, resolution, and reproducibility and demonstrated that it correlates with the level of firefly luciferase expression. Although the signal decreased gradually to about 20% of the initial value in the first month, the signal remained constant thereafter for more than 10 months. We demonstrated that the light signal can be used as a reporter by using a bicistronic vector. This is the first study to document noninvasive monitoring of long-term transgene expression in the adult mouse brain and provides the basis for applying BLI in the study of brain disease and gene therapeutic strategies.