From wires to waves, a novel sensor system for in vivo pressure monitoring.

Pressure monitoring in various organs of the body is essential for appropriate diagnostic and therapeutic purposes. In almost all situations, monitoring is performed in a hospital setting. Technological advances not only promise to improve clinical pressure monitoring systems, but also engage toward the development of fully implantable systems in ambulatory patients. Such systems would not only provide longitudinal time monitoring to healthcare personnel, but also to the patient who could adjust their way-of-life in response to the measurements. In the past years, we have developed a new type of piezoresistive pressure sensor system. Different bench tests have demonstrated that it delivers precise and reliable pressure measurements in real-time. The potential of this system was confirmed by a continuous recording in a patient that lasted for almost a day. In the present study, we further characterized the functionality of this sensor system by conducting in vivo implantation experiments in nine female farm pigs. To get a step closer to a fully implantable system, we also adapted two different wireless communication solutions to the sensor system. The communication protocols are based on MICS (Medical Implant Communication System) and BLE (Bluetooth Low Energy) communication. As a proof-of-concept, implantation experiments in nine female pigs demonstrated the functionality of both systems, with a notable technical superiority of the BLE.

Integrated software for multi-dimensional analysis of motion using tracking, electrophysiology, and sensor signals.

Tracking followed by analysis of specific point-of-interest from conventional or high-speed video recordings have been widely used for decades in various scientific disciplines such as sport, physiotherapy, and behavioral science. Another method used to characterize movement in 3D involves the use of motion capture systems, which produce files containing a collection of 3D-coordinates and corresponding timestamps. When studying animal or human movement, combining motion tracking with other recording methods-like monitoring muscle activity or sensor signals-can yield valuable insights. However, manual analysis of data from these diverse sources can be time-consuming and prone to errors. To address this issue, this article introduces a new, free, and open-source software developed in MATLAB. This software can be used as-is, or developed further to meet specific requirements. Once the coordinates are imported, multiple tools can be used for data preprocessing, such as to correct mistakes that may have occurred during tracking because of software errors or suboptimal video quality. In addition, the software can import coordinates from multiple cameras and combine them into a unified data series. With these inputs, the software can automatically calculate kinematic parameters and descriptive statistics, generate 2D and 3D animations, and analyze gait cycles, enabling swift and accurate analysis of multidimensional motion data. Moreover, the software can import electrophysiology traces and sensor signals, which can be filtered, rectified, smoothed, and correlated with the kinematic data in various ways. Thanks to its user-friendly graphical user interface, the software is easy to navigate and can be used to analyze complex movements without any need for coding skills. This versatile tool is well-suited for a wide range of experimental contexts, making it a valuable resource for researchers across diverse scientific disciplines.

How to generate graded spinal cord injuries in swine - tools and procedures.

Spinal cord injury (SCI) is a medically, psychologically and socially disabling condition. A large body of our knowledge on the basic mechanisms of SCI has been gathered in rodents. For preclinical validation of promising therapies, the use of animal models that are closer to humans has several advantages. This has promoted the more-intensive development of large-animal models for SCI during the past decade. We recently developed a multimodal SCI apparatus for large animals that generated biomechanically reproducible impacts in vivo. It is composed of a spring-load impactor and support systems for the spinal cord and the vertebral column. We now present the functional outcome of farm pigs and minipigs injured with different lesion strengths. There was a correlation between the biomechanical characteristics of the impact, the functional outcome and the tissue damage observed several weeks after injury. We also provide a detailed description of the procedure to generate such a SCI in both farm pigs and minipigs, in the hope to ease the adoption of the swine model by other research groups.

Author Correction: Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors.

In vivo Cell Tracking Using Non-invasive Imaging of Iron Oxide-Based Particles with Particular Relevance for Stem Cell-Based Treatments of Neurological and Cardiac Disease.

Stem cell-based therapeutics is a rapidly developing field associated with a number of clinical challenges. One such challenge lies in the implementation of methods to track stem cells and stem cell-derived cells in experimental animal models and in the living patient. Here, we provide an overview of cell tracking in the context of cardiac and neurological disease, focusing on the use of iron oxide-based particles (IOPs) visualized in vivo using magnetic resonance imaging (MRI). We discuss the types of IOPs available for such tracking, their advantages and limitations, approaches for labeling cells with IOPs, biological interactions and effects of IOPs at the molecular and cellular levels, and MRI-based and associated approaches for in vivo and histological visualization. We conclude with reviews of the literature on IOP-based cell tracking in cardiac and neurological disease, covering both preclinical and clinical studies.

Development of a Multimodal Apparatus to Generate Biomechanically Reproducible Spinal Cord Injuries in Large Animals.

Rodents are widespread animal models in spinal cord injury (SCI) research. They have contributed to obtaining important information. However, some treatments only tested in rodents did not prove efficient in clinical trials. This is probably a result of significant differences in the physiology, anatomy, and complexity between humans and rodents. To bridge this gap in a better way, a few research groups use pig models for SCI. Here we report the development of an apparatus to perform biomechanically reproducible SCI in large animals, including pigs. We present the iterative process of engineering, starting with a weight-drop system to ultimately produce a spring-load impactor. This device allows a graded combination of a contusion and a compression injury. We further engineered a device to entrap the spinal cord and prevent it from escaping at the moment of the impact. In addition, it provides identical resistance around the cord, thereby, optimizing the inter-animal reproducibility. We also present other tools to straighten the vertebral column and to ease the surgery. Sensors mounted on the impactor provide information to assess the inter-animal reproducibility of the impacts. Further evaluation of the injury strength using neurophysiological recordings, MRI scans, and histology shows consistency between impacts. We conclude that this apparatus provides biomechanically reproducible spinal cord injuries in pigs.

Locomotor central pattern generator excitability states and serotonin sensitivity after spontaneous recovery from a neonatal lumbar spinal cord injury.

The spinal locomotor central pattern generator (CPG) in neonatal mice exhibits diverse output patterns, ranging from sub-rhythmic to multi-rhythmic to fictive locomotion, depending on its general level of excitation and neuromodulatory status. We have recently reported that the locomotor CPG in neonatal mice rapidly recovers the ability to produce neurochemically induced fictive locomotion following an upper lumbar spinal cord compression injury. Here we address the question of recovery of multi-rhythmic activity and the serotonin-sensitivity of the CPG. In isolated spinal cords from control and 3 days post-injury mice, application of dopamine and NMDA elicited multi-rhythmic activity with slow and fast components. The slow component comprised 10-20 s episodes of activity that were synchronous in ipsilateral or all lumbar ventral roots, and the fast components involved bursts within these episodes that displayed coordinated patterns of alternation between ipsilateral roots. Rhythm strength was the same in control and injured spinal cords. However, power spectral analysis of signal within episodes showed a reduced peak frequency after recovery. In control spinal cords, serotonin triggered fictive locomotion only when applied at high concentration (30 µM, constant NMDA). By contrast, in about 50% of injured preparations fictive locomotion was evoked by 2-3 times lower serotonin concentrations (10-15 µM). This increased serotonin sensitivity was correlated with post-injury changes in the expression of specific serotonin receptor transcripts, but not of dopamine receptor transcripts.

Rapid recovery and altered neurochemical dependence of locomotor central pattern generation following lumbar neonatal spinal cord injury.

KEY POINTS: Spinal compression injury targeted to the neonatal upper lumbar spinal cord, the region of highest hindlimb locomotor rhythmogenicity, leads to an initial paralysis of the hindlimbs. Behavioural recovery is evident within a few days and approaches normal function within about 3 weeks. Fictive locomotion in the isolated injured spinal cord cannot be elicited by a neurochemical cocktail containing NMDA, dopamine and serotonin 1 day post-injury, but can 3 days post-injury as readily as in the uninjured spinal cord. Low frequency coordinated rhythmic activity can be elicited in the isolated uninjured spinal cord by NMDA + dopamine (without serotonin), but not in the isolated injured spinal cord. In both the injured and uninjured spinal cord, eliciting bona fide fictive locomotion requires the additional presence of serotonin. ABSTRACT: Following incomplete compression injury in the thoracic spinal cord of neonatal mice 1 day after birth (P1), we previously reported that virtually normal hindlimb locomotor function is recovered within about 3 weeks despite substantial permanent thoracic tissue loss. Here, we asked whether similar recovery occurs following lumbar injury that impacts more directly on the locomotor central pattern generator (CPG). As in thoracic injuries, lumbar injuries caused about 90% neuronal loss at the injury site and increased serotonergic innervation below the injury. Motor recovery was slower after lumbar than thoracic injury, but virtually normal function was attained by P25 in both cases. Locomotor CPG status was tested by eliciting fictive locomotion in isolated spinal cords using a widely used neurochemical cocktail (NMDA, dopamine, serotonin). No fictive locomotion could be elicited 1 day post-injury, but could within 3 days post-injury as readily as in age-matched uninjured control spinal cords. Burst patterning and coordination were largely similar in injured and control spinal cords but there were differences. Notably, in both groups there were two main locomotor frequencies, but injured spinal cords exhibited a shift towards the higher frequency. Injury also altered the neurochemical dependence of locomotor CPG output, such that injured spinal cords, unlike control spinal cords, were incapable of generating low frequency rhythmic coordinated activity in the presence of NMDA and dopamine alone. Thus, the neonatal spinal cord also exhibits remarkable functional recovery after lumbar injuries, but the neurochemical sensitivity of locomotor circuitry is modified in the process.

Cellular reactions and compensatory tissue re-organization during spontaneous recovery after spinal cord injury in neonatal mice.

Following incomplete spinal cord injuries, neonatal mammals display a remarkable degree of behavioral recovery. Previously, we have demonstrated in neonatal mice a wholesale re-establishment and reorganization of synaptic connections from some descending axon tracts (Boulland et al.: PLoS One 8 (2013)). To assess the potential cellular mechanisms contributing to this recovery, we have here characterized a variety of cellular sequelae following thoracic compression injuries, focusing particularly on cell loss and proliferation, inflammation and reactive gliosis, and the dynamics of specific types of synaptic terminals. Early during the period of recovery, regressive events dominated. Tissue loss near the injury was severe, with about 80% loss of neurons and a similar loss of axons that later make up the white matter. There was no sign of neurogenesis, no substantial astroglial or microglial proliferation, no change in the ratio of M1 and M2 microglia and no appreciable generation of the terminal complement peptide C5a. One day after injury the number of synaptic terminals on lumbar motoneurons had dropped by a factor of 2, but normalized by 6 days. The ratio of VGLUT1/2+ to VGAT+ terminals remained similar in injured and uninjured spinal cords during this period. By 24 days after injury, when functional recovery is nearly complete, the density of 5-HT+ fibers below the injury site had increased by a factor of 2.5. Altogether this study shows that cellular reactions are diverse and dynamic. Pronounced recovery of both excitatory and inhibitory terminals and an increase in serotonergic innervation below the injury, coupled with a general lack of inflammation and reactive gliosis, are likely to contribute to the recovery. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 928-946, 2017.

A Neonatal Mouse Spinal Cord Compression Injury Model.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life(1), this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques(1). Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections(1).

Loss of Projections, Functional Compensation, and Residual Deficits in the Mammalian Vestibulospinal System of Hoxb1-Deficient Mice.

The genetic mechanisms underlying the developmental and functional specification of brainstem projection neurons are poorly understood. Here, we use transgenic mouse tools to investigate the role of the gene Hoxb1 in the developmental patterning of vestibular projection neurons, with particular focus on the lateral vestibulospinal tract (LVST). The LVST is the principal pathway that conveys vestibular information to limb-related spinal motor circuits and arose early during vertebrate evolution. We show that the segmental hindbrain expression domain uniquely defined by the rhombomere 4 (r4) Hoxb1 enhancer is the origin of essentially all LVST neurons, but also gives rise to subpopulations of contralateral medial vestibulospinal tract (cMVST) neurons, vestibulo-ocular neurons, and reticulospinal (RS) neurons. In newborn mice homozygous for a Hoxb1-null mutation, the r4-derived LVST and cMVST subpopulations fail to form and the r4-derived RS neurons are depleted. Several general motor skills appear unimpaired, but hindlimb vestibulospinal reflexes, which are mediated by the LVST, are greatly reduced. This functional deficit recovers, however, during the second postnatal week, indicating a substantial compensation for the missing LVST. Despite the compensatory plasticity in balance, adult Hoxb1-null mice exhibit other behavioral deficits that manifest particularly in proprioception and interlimb coordination during locomotor tasks. Our results provide a comprehensive account of the developmental role of Hoxb1 in patterning the vestibular system and evidence for a remarkable developmental plasticity in the descending control of reflex limb movements. They also suggest an involvement of the lateral vestibulospinal tract in proprioception and in ensuring limb alternation generated by locomotor circuitry.

Reorganization of supramammillary-hippocampal pathways in the rat pilocarpine model of temporal lobe epilepsy: evidence for axon terminal sprouting.

In mesial temporal lobe epilepsy (MTLE), spontaneous seizures likely originate from a multi-structural epileptogenic zone, including several regions of the limbic system connected to the hippocampal formation. In this study, we investigate the structural connectivity between the supramammillary nucleus (SuM) and the dentate gyrus (DG) in the model of MTLE induced by pilocarpine in the rat. This hypothalamic nucleus, which provides major extracortical projections to the hippocampal formation, plays a key role in the regulation of several hippocampus-dependent activities, including theta rhythms, memory function and emotional behavior, such as stress and anxiety, functions that are known to be altered in MTLE. Our findings demonstrate a marked reorganization of DG afferents originating from the SuM in pilocarpine-treated rats. This reorganization, which starts during the latent period, is massive when animals become epileptic and continue to evolve during epilepsy. It is characterized by an aberrant distribution and an increased number of axon terminals from neurons of both lateral and medial regions of the SuM, invading the entire inner molecular layer of the DG. This reorganization, which reflects an axon terminal sprouting from SuM neurons, could contribute to trigger spontaneous seizures within an altered hippocampal intrinsic circuitry.

A neonatal mouse spinal cord injury model for assessing post-injury adaptive plasticity and human stem cell integration.

Despite limited regeneration capacity, partial injuries to the adult mammalian spinal cord can elicit variable degrees of functional recovery, mediated at least in part by reorganization of neuronal circuitry. Underlying mechanisms are believed to include synaptic plasticity and collateral sprouting of spared axons. Because plasticity is higher in young animals, we developed a spinal cord compression (SCC) injury model in the neonatal mouse to gain insight into the potential for reorganization during early life. The model provides a platform for high-throughput assessment of functional synaptic connectivity that is also suitable for testing the functional integration of human stem and progenitor cell-derived neurons being considered for clinical cell replacement strategies. SCC was generated at T9-T11 and functional recovery was assessed using an integrated approach including video kinematics, histology, tract tracing, electrophysiology, and high-throughput optical recording of descending inputs to identified spinal neurons. Dramatic degeneration of axons and synaptic contacts was evident within 24 hours of SCC, and loss of neurons in the injured segment was evident for at least a month thereafter. Initial hindlimb paralysis was paralleled by a loss of descending inputs to lumbar motoneurons. Within 4 days of SCC and progressively thereafter, hindlimb motility began to be restored and descending inputs reappeared, but with examples of atypical synaptic connections indicating a reorganization of circuitry. One to two weeks after SCC, hindlimb motility approached sham control levels, and weight-bearing locomotion was virtually indistinguishable in SCC and sham control mice. Genetically labeled human fetal neural progenitor cells injected into the injured spinal cord survived for at least a month, integrated into the host tissue and began to differentiate morphologically. This integrative neonatal mouse model provides opportunities to explore early adaptive plasticity mechanisms underlying functional recovery as well as the capacity for human stem cell-derived neurons to integrate functionally into spinal circuits.

Inhibition of tumor formation and redirected differentiation of glioblastoma cells in a xenotypic embryonic environment.

BACKGROUND: Tissue microenvironment plays key roles in regulating the progression of aggressive tumors. Tumors are uncommon in the early embryo, suggesting that embryonic tissue microenvironments are nonpermissive for tumors. Yet, the effects of embryonic tissue microenvironments on tumor cells have not been extensively studied. We have, therefore, tested the behavior of human glioblastoma multiforme (GBM) cells transplanted into a central neural tissue microenvironment in the chicken embryo. RESULTS: GBM cells were cultured as spheres to enrich for GBM stem cells (GSCs) and transduced with GFP for identification. Within the proliferative embryonic neural tissue, GSC-enriched GBM cells exhibited reduced proliferation and survival, altered gene expression, and formed no tumors, in marked contrast to their aggressive behavior in vitro and tumor formation in other tissue microenvironments including the chorioallantoic membrane of the chicken embryo and the brain of adult severe combined immunodeficiency (SCID) mice. Surviving cells in the spinal neural tube exhibited tumor-atypical expression profiles of neuron-, glia-, stem cell-, and tumor-related genes. CONCLUSIONS: Embryonic neural tissue provides a poor environment for GBM cell survival and tumor formation, and redirects differentiation toward a more benign phenotype. Understanding the anti-tumorigenic effects of this embryonic tissue microenvironment could provide opportunities to develop novel therapies for GBM treatment.

Epigenetic regulation of nestin expression during neurogenic differentiation of adipose tissue stem cells.

Adipose-tissue-derived stem cells (ASCs) have received considerable attention due to their easy access, expansion potential, and differentiation capacity. ASCs are believed to have the potential to differentiate into neurons. However, the mechanisms by which this may occur remain largely unknown. Here, we show that culturing ASCs under active proliferation conditions greatly improves their propensity to differentiate toward osteogenic, adipogenic, and neurogenic lineages. Neurogenic-induced ASCs express early neurogenic genes as well as markers of mature neurons, including voltage-gated ion channels. Nestin, highly expressed in neural progenitors, is upregulated by mitogenic stimulation of ASCs, and as in neural progenitors, then repressed during neurogenic differentiation. Nestin gene (NES) expression under these conditions appears to be regulated by epigenetic mechanisms. The neural-specific, but not muscle-specific, enhancer regions of NES are DNA demethylated by mitogenic stimulation, and remethylated upon neurogenic differentiation. We observe dynamic changes in histone H3K4, H3K9, and H3K27 methylation on the NES locus before and during neurogenic differentiation that are consistent with epigenetic processes involved in the regulation of NES expression. We suggest that ASCs are epigenetically prepatterned to differentiate toward a neural lineage and that this prepatterning is enhanced by demethylation of critical NES enhancer elements upon mitogenic stimulation preceding neurogenic differentiation. Our findings provide molecular evidence that the differentiation repertoire of ASCs may extend beyond mesodermal lineages.

Evaluation of intracellular labeling with micron-sized particles of iron oxide (MPIOs) as a general tool for in vitro and in vivo tracking of human stem and progenitor cells.

Magnetic resonance imaging (MRI)-based tracking is increasingly attracting attention as a means of better understanding stem cell dynamics in vivo. Intracellular labeling with micrometer-sized particles of iron oxide (MPIOs) provides a practical MRI-based approach due to superior detectability relative to smaller iron oxide particles. However, insufficient information is available about the general utility across cell types and the effects on cell vitality of MPIO labeling of human stem cells. We labeled six human cell types from different sources: mesenchymal stem cells derived from bone marrow (MSCs), mesenchymal stem cells derived from adipose tissue (ASCs), presumptive adult neural stem cells (ad-NSCs), fetal neural progenitor cells (f-NPCs), a glioma cell line (U87), and glioblastoma tumor stem cells (GSCs), with two different sizes of MPIOs (0.9 and 2.84 µm). Labeling and uptake efficiencies were highly variable among cell types. Several parameters of general cell function were tested in vitro. Only minor differences were found between labeled and unlabeled cells with respect to proliferation rate, mitotic duration, random motility, and capacity for differentiation to specific phenotypes. In vivo behavior was tested in chicken embryos and severe combined immunodeficient (SCID) mice. Postmortem histology showed that labeled cells survived and could integrate into various tissues. MRI-based tracking over several weeks in the SCID mice showed that labeled GSCs and f-NPCs injected into the brain exhibited translocations similar to those seen for unlabeled cells and as expected from migratory behavior described in previous studies. The results support MPIO-based cell tracking as a generally useful tool for studies of human stem cell dynamics in vivo.

Ontogenetic changes in the distribution of the vesicular GABA transporter VGAT correlate with the excitation/inhibition shift of GABA action.

GABA is the major inhibitory neurotransmitter in the adult CNS and is among others involved in the synchronization of large neuronal networks. During development, GABA acts as a morphogenetic factor and has transient excitatory actions in many brain regions. One distinct protein, the vesicular GABA transporter (VGAT), has been identified accumulating GABA into presynaptic vesicles prior to its exocytotic release. The function of VGAT and its distribution is well defined in the adult, but its contribution to the transient excitatory action at putative GABAergic nerve terminals in the immature brain and its potential roles in putative glutamatergic nerve terminals remain elusive. We have studied VGAT expression in the brain from late embryonic stages through several postnatal stages until adulthood. Quantitative immunoblotting and immunolabeling of tissue sections at the light microscope and the electron microscope levels show an abrupt augmentation in VGAT staining in the cerebral cortex during the first three postnatal weeks, resembling the increase in other proteins involved in GABA synthesis and recycling in the same time frame - such as GAD65, GAD67, GAT1 (Slc6a1) and SN1 (Slc38a3) - and coincides with the synaptogenetic spurt. Dynamic changes in the expression of VGAT are seen in many cellular populations and in several layers in different brain regions. However, mossy fiber terminals (MFT) elude staining for VGAT. We also demonstrate that VGAT(+) nerve terminals undergo a developmental reorganization so that from targeting primarily the dendrites of the principal neurons in several brain regions in the immature brain, they target the soma of the same cells in the adult. This shift in the targeted subcellular compartment coincides with the conversion of the chloride gradient across neuronal membranes and suggests that it may be important for the shift of GABA action from excitation to inhibition and for the establishment of the potent synchronization of neuronal networks.