Pharmacological Rescue of Dendritic Pathology in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is a genetic pathology characterized by brain hypotrophy and severe cognitive disability. Although defective neurogenesis is an important determinant of cognitive impairment, a severe dendritic pathology appears to be an equally important factor. It is well established that serotonin plays a pivotal role both on neurogenesis and dendritic maturation. Since the serotonergic system is profoundly altered in the DS brain, we wondered whether defects in the hippocampal development can be rescued by treatment with fluoxetine, a selective serotonin reuptake inhibitor and a widely used antidepressant drug. A previous study of our group showed that fluoxetine fully restores neurogenesis in the Ts65Dn mouse model of DS and that this effect is accompanied by a recovery of memory functions. The goal of the current study was to establish whether fluoxetine also restores dendritic development and maturation. In mice aged 45 days, treated with fluoxetine in the postnatal period P3-P15, we examined the dendritic arbor of newborn and mature granule cells of the dentate gyrus (DG). The granule cells of trisomic mice had a severely hypotrophic dendritic arbor, fewer spines and a reduced innervation than euploid mice. Treatment with fluoxetine fully restored all these defects. Moreover the impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons was fully normalized in treated trisomic mice, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The widespread beneficial effects of fluoxetine on the hippocampal formation suggest that early treatment with fluoxetine can be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions. These findings may open the way for future clinical trials in children and adolescents with DS.

Combining 3D tracking and surgical instrumentation to determine the stiffness of spinal motion segments: a validation study

The spine is a complex structure that provides motion in three directions: flexion and extension, lateral bending and axial rotation. So far, the investigation of the mechanical and kinematic behavior of the basic unit of the spine, a motion segment, is predominantly a domain of in vitro experiments on spinal loading simulators. Most existing approaches to measure spinal stiffness intraoperatively in an in vivo environment use a distractor. However, these concepts usually assume a planar loading and motion. The objective of our study was to develop and validate an apparatus, that allows to perform intraoperative in vivo measurements to determine both the applied force and the resulting motion in three dimensional space. The proposed setup combines force measurement with an instrumented distractor and motion tracking with an optoelectronic system. As the orientation of the applied force and the three dimensional motion is known, not only force-displacement, but also moment-angle relations could be determined. The validation was performed using three cadaveric lumbar ovine spines. The lateral bending stiffness of two motion segments per specimen was determined with the proposed concept and compared with the stiffness acquired on a spinal loading simulator which was considered to be gold standard. The mean values of the stiffness computed with the proposed concept were within a range of ±15% compared to data obtained with the spinal loading simulator under applied loads of less than 5 Nm.

Microfenestration using the CUSA Excel ultrasonic aspiration system in chondrodystrophic dogs with thoracolumbar disk extrusion: a descriptive cadaveric and clinical study

OBJECTIVE: To describe an ultrasonic surgical aspirator assisted disk fenestration technique in dogs. STUDY DESIGN: Descriptive cadaveric and prospective clinical study. ANIMALS: Fresh Beagle cadavers (n=5) and 10 chondrodystrophic dogs with thoracolumbar disk extrusion. METHODS: Cadaveric study: Intervertebral disks T12-L2 were fenestrated with the CUSA Excel in 5 Beagle cadavers, and fenestration efficacy assessed by morphologic examination of the completeness of fenestration and size of annulotomy. Clinical study: the affected intervertebral disk was fenestrated in 10 chondrodystrophic dogs treated by hemilaminectomy for thoracolumbar disk disease. Efficacy of fenestration was evaluated. RESULTS: Mean time necessary to perform CUSA assisted fenestration was 8 minutes (range, 5-10 minutes) for each disk in cadavers and patients. In cadaver spines, removal of the nucleus pulposus was complete in 11/15 disks. In 4 disks, remnants of nucleus pulposus material were observed on the contralateral side. Nuclear material was normal in 9/15 disks and showed evidence of chondroid degeneration on histopathologic examination in the 6 disks. Median annulotomy size was 3 mm. Clinically, no signs of early recurrence were observed and all dogs recovered uneventfully. CONCLUSIONS: CUSA assisted fenestration is a safe and efficient method of fenestration for removal of most of the nucleus pulposus through a limited annulotomy.

Influence of Partial Lateral Corpectomy with and without Hemilaminectomy on Canine Thoracolumbar Stability: A Biomechanical Study

OBJECTIVE: To analyze the biomechanical changes induced by partial lateral corpectomy (PLC) and a combination of PLC and hemilaminectomy in a T13-L3 spinal segment in nonchondrodystrophic dogs. STUDY DESIGN: In vitro biomechanical cadaveric study. SAMPLE POPULATION: T13-L3 spinal segments (n = 10) of nonchondrodystrophic dogs (weighing, 25-38 kg). METHODS: A computed tomography (CT) scan of each T13-L3 spinal segment was performed. A loading simulator for flexibility analysis was used to determine the range of motion (ROM) and neutral zone (NZ) during flexion/extension, lateral bending, and axial rotation. A servohydraulic testing machine was used to determine the changes in stiffness during compression, dorsoventral, and lateral shear. All spines were tested intact, after PLC in the left intervertebral space of L1-L2, and after a combination of PLC and hemilaminectomy. RESULTS: Statistically significant increases in ROM and NZ (P < .05) were detected during flexion/extension and lateral bending when PLC was performed. A significant increase in ROM (P < .001) was noted during axial rotation and flexion after PLC and hemilaminectomy. Stiffness decreased significantly during compression and dorsoventral shear after each procedure. Decreased stiffness during lateral shear was only significant after a combination of both procedures. CONCLUSION: PLC might lead to some spinal instability; these changes are enhanced when a hemilaminectomy is added.

Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing

Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high-pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine-structural changes at hippocampal mossy fiber synapses associated with chemically induced long-term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP-induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13-1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre- and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM.

Spine Ca2+ signaling in spike-timing-dependent plasticity

Calcium is a second messenger, which can trigger the modification of synaptic efficacy. We investigated the question of whether a differential rise in postsynaptic Ca2+ ([Ca2+]i) alone is sufficient to account for the induction of long-term potentiation (LTP) and long-term depression (LTD) of EPSPs in the basal dendrites of layer 2/3 pyramidal neurons of the somatosensory cortex. Volume-averaged [Ca2+]i transients were measured in spines of the basal dendritic arbor for spike-timing-dependent plasticity induction protocols. The rise in [Ca2+]i was uncorrelated to the direction of the change in synaptic efficacy, because several pairing protocols evoked similar spine [Ca2+]i transients but resulted in either LTP or LTD. The sequence dependence of near-coincident presynaptic and postsynaptic activity on the direction of changes in synaptic strength suggested that LTP and LTD were induced by two processes, which were controlled separately by postsynaptic [Ca2+]i levels. Activation of voltage-dependent Ca2+ channels before metabotropic glutamate receptors (mGluRs) resulted in the phospholipase C-dependent (PLC-dependent) synthesis of endocannabinoids, which acted as a retrograde messenger to induce LTD. LTP required a large [Ca2+]i transient evoked by NMDA receptor activation. Blocking mGluRs abolished the induction of LTD and uncovered the Ca2+-dependent induction of LTP. We conclude that the volume-averaged peak elevation of [Ca2+]i in spines of layer 2/3 pyramids determines the magnitude of long-term changes in synaptic efficacy. The direction of the change is controlled, however, via a mGluR-coupled signaling cascade. mGluRs act in conjunction with PLC as sequence-sensitive coincidence detectors when postsynaptic precede presynaptic action potentials to induce LTD. Thus presumably two different Ca2+ sensors in spines control the induction of spike-timing-dependent synaptic plasticity.

Osseointegration of hollow cylinder based spinal implants in normal and osteoporotic vertebrae: a sheep study

INTRODUCTION: Osteoporosis is not only responsible for an increased number of metaphyseal and spinal fractures but it also complicates their treatment. To prevent the initial loosening, we developed a new implant with an enlarged implant/bone interface based on the concept of perforated, hollow cylinders. We evaluated whether osseointegration of a hollow cylinder based implant takes place in normal or osteoporotic bone of sheep under functional loading conditions during anterior stabilization of the lumbar spine. MATERIALS AND METHODS: Osseointegration of the cylinders and status of the fused segments (ventral corpectomy, replacement with iliac strut, and fixation with testing implant) were investigated in six osteoporotic (age 6.9 +/- 0.8 years, mean body weight 61.1 +/- 5.2 kg) and seven control sheep (age 6.1 +/- 0.2 years, mean body weight 64.9 +/- 5.7 kg). Osteoporosis was introduced using a combination protocol of ovariectomy, high-dose prednisone, calcium and phosphor reduced diet and movement restriction. Osseointegration was quantified using fluorescence and conventional histology; fusion status was determined using biomechanical testing of the stabilized segment in a six-degree-of-freedom loading device as well as with radiological and histological staging. RESULTS: Intact bone trabeculae were found in 70% of all perforations without differences between the two groups (P = 0.26). Inside the cylinders, bone volume/total volume was significantly higher than in the control vertebra (50 +/- 16 vs. 28 +/- 13%) of the same animal (P<0.01), but significantly less (P<0.01) than in the near surrounding (60 +/- 21%). After biomechanical testing as described in Sect. "Materials and methods", seven spines (three healthy and four osteoporotic) were classified as completely fused and six (four healthy and two osteoporotic) as not fused after a 4-month observation time. All endplates were bridged with intact trabeculae in the histological slices. CONCLUSIONS: The high number of perforations, filled with intact trabeculae, indicates an adequate fixation; bridging trabeculae between adjacent endplates and tricortical iliac struts in all vertebrae indicates that the anchorage is adequate to promote fusion in this animal model, even in the osteoporotic sheep.

Calcium indicator loading of neurons using single-cell electroporation

Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.

New ways of looking at synapses

Current concepts of synaptic fine-structure are derived from electron microscopic studies of tissue fixed by chemical fixation using aldehydes. However, chemical fixation with glutaraldehyde and paraformaldehyde and subsequent dehydration in ethanol result in uncontrolled tissue shrinkage. While electron microscopy allows for the unequivocal identification of synaptic contacts, it cannot be used for real-time analysis of structural changes at synapses. For the latter purpose advanced fluorescence microscopy techniques are to be applied which, however, do not allow for the identification of synaptic contacts. Here, two approaches are described that may overcome, at least in part, some of these drawbacks in the study of synapses. By focusing on a characteristic, easily identifiable synapse, the mossy fiber synapse in the hippocampus, we first describe high-pressure freezing of fresh tissue as a method that may be applied to study subtle changes in synaptic ultrastructure associated with functional synaptic plasticity. Next, we propose to label presynaptic mossy fiber terminals and postsynaptic complex spines on CA3 pyramidal neurons by different fluorescent dyes to allow for the real-time monitoring of these synapses in living tissue over extended periods of time. We expect these approaches to lead to new insights into the structure and function of central synapses.

The effect of pulsed jet lavage in vertebroplasty on injection forces of polymethylmethacrylate bone cement, material distribution, and potential fat embolism: a cadaver study

STUDY DESIGN: In vitro testing of vertebroplasty techniques including pulsed jet-lavage for fat and marrow removal in human cadaveric lumbar and thoracic vertebrae. OBJECTIVE: To develop jet-lavage techniques for vertebroplasty and investigate their effect on cement distribution, injection forces, and fat embolism. SUMMARY OF BACKGROUND DATA: The main complications of cement vertebroplasty are cement leakage and pulmonary fat embolism, which can have fatal consequences and are difficult to prevent reliably by current vertebroplasty techniques. METHODS: Twenty-four vertebrae (Th8-L04) from 5 osteoporotic cadaver spines were grouped in triplets depending on bone mineral density (BMD). Before polymethylmethacrylate (PMMA) vertebroplasty, a pulsatile jet-lavage for removal of intertrabecular fat and bone marrow was performed in 2 groups with 8 specimens each, performing radial and axial irrigation from the biopsy needles. One hundred mL of Ringer solution were injected through 1 pedicle and regained by low vacuum via the contralateral pedicle. Eight control vertebrae were not irrigated. All specimens underwent standardized PMMA cement augmentation injecting 20% of the vertebral volume. Injection forces, cement distribution, and extravasations were quantified. RESULTS: All irrigation solution could be retrieved with the vacuum applied. A Kruskal-Wallis test revealed significantly higher injection forces of the control group as compared with the irrigated groups (P = 0.021). Dilatation of the syringe at forces above 300 N occurred in 75% of the untreated compared with 12.5% of the lavaged specimens. CT distribution analysis showed more homogenous cement distribution of the cement and significantly less extravasation in the irrigated specimens. CONCLUSION: The developed lavage technique for vertebroplasty showed to be feasible and reproducible. The reduction of injection forces would allow the use of more viscous PMMA cement lowering the risk for cement embolization and results in a safer procedure. The wash-out of bone marrow and the possible reduction of pulmonary fat embolism have to be verified with in vivo models.

Evaluation of early tissue reactions after lumbar intertransverse process fusion using CT in a rabbit

OBJECTIVE: The objective of the study was to evaluate tissue reactions such as bone genesis, cartilage genesis and graft materials in the early phase of lumbar intertransverse process fusion in a rabbit model using computed tomography (CT) imaging with CT intensity (Hounsfield units) measurement, and to compare these data with histological results. MATERIALS AND METHODS: Lumbar intertransverse process fusion was performed on 18 rabbits. Four graft materials were used: autograft bone (n = 3); collagen membrane soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) (n = 5); granular calcium phosphate (n = 5); and granular calcium phosphate coated with rhBMP-2 (n = 5). All rabbits were euthanized 3 weeks post-operatively and lumbar spines were removed for CT imaging and histological examination. RESULTS: Computed tomography imaging demonstrated that each fusion mass component had the appropriate CT intensity range. CT also showed the different distributions and intensities of bone genesis in the fusion masses between the groups. Each component of tissue reactions was identified successfully on CT images using the CT intensity difference. Using CT color mapping, these observations could be easily visualized, and the results correlated well with histological findings. CONCLUSIONS: The use of CT intensity is an effective approach for observing and comparing early tissue reactions such as newly synthesized bone, newly synthesized cartilage, and graft materials after lumbar intertransverse process fusion in a rabbit model.

Biomechanical analysis of torsion and shear forces in lumbar and lumbosacral spine segments of nonchondrodystrophic dogs

OBJECTIVE: To determine stiffness and load-displacement curves as a biomechanical response to applied torsion and shear forces in cadaveric canine lumbar and lumbosacral specimens. STUDY DESIGN: Biomechanical study. ANIMALS: Caudal lumbar and lumbosacral functional spine units (FSU) of nonchondrodystrophic large-breed dogs (n=31) with radiographically normal spines. METHODS: FSU from dogs without musculoskeletal disease were tested in torsion in a custom-built spine loading simulator with 6 degrees of freedom, which uses orthogonally mounted electric motors to apply pure axial rotation. For shear tests, specimens were mounted to a custom-made shear-testing device, driven by a servo hydraulic testing machine. Load-displacement curves were recorded for torsion and shear. RESULTS: Left and right torsion stiffness was not different within each FSU level; however, torsional stiffness of L7-S1 was significantly smaller compared with lumbar FSU (L4-5-L6-7). Ventral/dorsal stiffness was significantly different from lateral stiffness within an individual FSU level for L5-6, L6-7, and L7-S1 but not for L4-5. When the data from 4 tested shear directions from the same specimen were pooled, level L5-6 was significantly stiffer than L7-S1. CONCLUSIONS: Increased range of motion of the lumbosacral joint is reflected by an overall decreased shear and rotational stiffness at the lumbosacral FSU. CLINICAL RELEVANCE: Data from dogs with disc degeneration have to be collected, analyzed, and compared with results from our chondrodystrophic large-breed dogs with radiographically normal spines.

Modeling Calcium Concentration and Biochemical Reactions

Almost all regions of the brain receive one or more neuromodulatory inputs, and disrupting these inputs produces deficits in neuronal function. Neuromodulators act through intracellular second messenger pathways to influence the electrical properties of neurons, integration of synaptic inputs, spatio-temporal firing dynamics of neuronal networks, and, ultimately, systems behavior. Second messengers pathways consist of series of bimolecular reactions, enzymatic reactions, and diffusion. Calcium is the second messenger molecule with the most effectors, and thus is highly regulated by buffers, pumps and intracellular stores. Computational modeling provides an innovative, yet practical method to evaluate the spatial extent, time course and interaction among second messenger pathways, and the interaction of second messengers with neuron electrical properties. These processes occur both in compartments where the number of molecules are large enough to describe reactions deterministically (e.g. cell body), and in compartments where the number of molecules is small enough that reactions occur stochastically (e.g. spines). – In this tutorial, I explain how to develop models of second messenger pathways and calcium dynamics. The first part of the tutorial explains the equations used to model bimolecular reactions, enzyme reactions, calcium release channels, calcium pumps and diffusion. The second part explains some of the GENESIS, Kinetikit and Chemesis objects that implement the appropriate equations. In depth explanation of calcium and second messenger models is provided by reviewing code, both in XPP, Chemesis and Kinetikit, that implements simple models of calcium dynamics and second messenger cascades.

Augmentation of mechanical properties in osteoporotic vertebral bones--a biomechanical investigation of vertebroplasty efficacy with different bone cements

Recent clinical trials have reported favorable early results for transpedicular vertebral cement reinforcement of osteoporotic vertebral insufficiencies. There is, however, a lack of basic data on the application, safety and biomechanical efficacy of materials such as polymethyl-methacrylate (PMMA) and calciumphospate (CaP) cements. The present study analyzed 33 vertebral pairs from five human cadaver spines. Thirty-nine vertebrae were osteoporotic (bone mineral density < 0.75 g/cm2), 27 showed nearly normal values. The cranial vertebra of each pair was augmented with either PMMA (Palacos E-Flow) or experimental brushite cement (EBC), with the caudal vertebra as a control. PMMA and EBC were easy to inject, and vertebral fillings of 20-50% were achieved. The maximal possible filling was inversely correlated to the bone mineral density (BMD) values. Cement extrusion into the spinal canal was observed in 12% of cases. All specimens were subjected to axial compression tests in a displacement-controlled mode. From load-displacement curves, the stiffness, S, and the maximal force before failure, Fmax, were determined. Compared with the native control vertebrae, a statistically significant increase in vertebral stiffness and Fmax was observed by the augmentation. With PMMA the stiffness increased by 174% (P = 0.018) and Fmax by 195% (P = 0.001); the corresponding augmentation with EBC was 120% (P = 0.03) and 113% (P = 0.002). The lower the initial BMD, the more pronounced was the augmentation effect. Both PMMA and EBC augmentation reliably and significantly raised the stiffness and maximal tolerable force until failure in osteoporotic vertebral bodies. In non-porotic specimens, no significant increase was achieved.

Cellular dynamic simulator: an event driven molecular simulation environment for cellular physiology.

In this paper, we present the Cellular Dynamic Simulator (CDS) for simulating diffusion and chemical reactions within crowded molecular environments. CDS is based on a novel event driven algorithm specifically designed for precise calculation of the timing of collisions, reactions and other events for each individual molecule in the environment. Generic mesh based compartments allow the creation / importation of very simple or detailed cellular structures that exist in a 3D environment. Multiple levels of compartments and static obstacles can be used to create a dense environment to mimic cellular boundaries and the intracellular space. The CDS algorithm takes into account volume exclusion and molecular crowding that may impact signaling cascades in small sub-cellular compartments such as dendritic spines. With the CDS, we can simulate simple enzyme reactions; aggregation, channel transport, as well as highly complicated chemical reaction networks of both freely diffusing and membrane bound multi-protein complexes. Components of the CDS are generally defined such that the simulator can be applied to a wide range of environments in terms of scale and level of detail. Through an initialization GUI, a simple simulation environment can be created and populated within minutes yet is powerful enough to design complex 3D cellular architecture. The initialization tool allows visual confirmation of the environment construction prior to execution by the simulator. This paper describes the CDS algorithm, design implementation, and provides an overview of the types of features available and the utility of those features are highlighted in demonstrations.