Exosomes Isolated From Platelet-Rich Plasma and Mesenchymal Stem Cells Promote Recovery of Function After Muscle Injury.

BACKGROUND: Clinical use of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) has gained momentum as treatment for muscle injuries. Exosomes, or small cell-derived vesicles, could be helpful if they could deliver the same or better physiological effect without cell transplantation into the muscle. HYPOTHESIS: Local delivery of exosomes derived from PRP (PRP-exos) or MSCs (MSC-exos) to injured muscles hastens recovery of contractile function. STUDY DESIGN: Controlled laboratory study. METHODS: In a rat model, platelets were isolated from blood, and MSCs were isolated from bone marrow and expanded in culture; exosomes from both were isolated through ultracentrifugation. The tibialis anterior muscles were injured in vivo using maximal lengthening contractions. Muscles were injected with PRP-exos or MSC-exos (immediately after injury and 5 and 10 days after injury); controls received an equal volume of saline. Histological and biochemical analysis was performed on tissues for all groups. RESULTS: Injury resulted in a significant loss of maximal isometric torque (66% ± 3%) that gradually recovered over 2 weeks. Both PRP-exos and MSC-exos accelerated recovery, with similar faster recovery of contractile function over the saline-treated group at 5, 10, and 15 days after injury (P < .001). A significant increase in centrally nucleated fibers was seen with both types of exosome groups by day 15 (P < .01). Genes involved in skeletal muscle regeneration were modulated by different exosomes. Muscles treated with PRP-exos had increased expression of Myogenin gene (P < .05), whereas muscles treated with MSC-exos had reduced expression of TGF-ß (P < .05) at 10 days after muscle injury. CONCLUSION: Exosomes derived from PRP or MSCs can facilitate recovery after a muscle strain injury in a small-animal model likely because of factors that can modulate inflammation, fibrosis, and myogenesis. CLINICAL RELEVANCE: Given their small size, low immunogenicity, and ease with which they can be obtained, exosomes could represent a novel therapy for many orthopaedic ailments.

Magnetic Enhancement of Stem Cell-Targeted Delivery into the Brain Following MR-Guided Focused Ultrasound for Opening the Blood-Brain Barrier.

Focused ultrasound (FUS)-mediated blood-brain barrier disruption (BBBD) can enable even large therapeutics such as stem cells to enter the brain from the bloodstream. However, the efficiency is relatively low. Our previous study showed that human neural progenitor cells (hNPCs) loaded with superparamagnetic iron oxide nanoparticles (SPIONs) in culture were attracted by an external magnetic field. In vivo, enhanced brain retention was observed near a magnet mounted on the skull in a rat model of traumatic brain injury, where BBBD also occurs. The goal of the current study was to determine whether magnetic attraction of SPION-loaded hNPCs would also enhance their retention in the brain after FUS-mediated BBBD. A small animal magnetic resonance imaging (MRI)-guided FUS system operating at 1.5 MHz was used to treat rats (∼120 g) without tissue damage or hemorrhage. Evidence of successful BBBD was validated with both radiologic enhancement of gadolinium on postsonication TI MRI and whole brain section visualization of Evans blue dye. The procedure was then combined with the application of a powerful magnet to the head directly after intravenous injection of the hNPCs. Validation of cells within the brain was performed by staining with Perls' Prussian blue for iron and by immunohistochemistry with a human-specific antigen. By injecting equal numbers of iron oxide (SPIONs) and noniron oxide nanoparticles-loaded hNPCs, each labeled with a different fluorophore, we found significantly greater numbers of SPIONs-loaded cells retained in the brain at the site of BBBD as compared to noniron loaded cells. This result was most pronounced in regions of the brain closest to the skull (dorsal cortex) in proximity to the magnet surface. A more powerful magnet and a Halbach magnetic array resulted in more effective retention of SPION-labeled cells in even deeper brain regions such as the striatum and ventral cortex. There, up to 90% of hNPCs observed contained SPIONs compared to 60% to 70% with the less powerful magnet. Fewer cells were observed at 24 h posttreatment compared to 2 h (primarily in the dorsal cortex). These results demonstrate that magnetic attraction can substantially enhance the retention of stem cells after FUS-mediated BBBD. This procedure could provide a safer and less invasive approach for delivering stem cells to the brain, compared to direct intracranial injections, substantially reducing the risk of bleeding and infection.

SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd.

Cell-Based Therapy in TBI: Magnetic Retention of Neural Stem Cells In Vivo.

Stem cell therapy is under active investigation for traumatic brain injury (TBI). Noninvasive stem cell delivery is the preferred method, but retention of stem cells at the site of injury in TBI has proven challenging and impacts effectiveness. To investigate the effects of applying a magnetic field on cell homing and retention, we delivered human neuroprogenitor cells (hNPCs) labeled with a superparamagnetic nanoparticle into post-TBI animals in the presence of a static magnetic field. We have previously devised a method of loading hNPCs with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles Molday ION Rhodamine B (MIRB™). Labeling of hNPCs (MIRB-hNPCs) does not affect hNPC viability, proliferation, or differentiation. The 0.6 tesla (T) permanent magnet was placed ∼4 mm above the injured parietal cortex prior to intracarotid injection of 4 × 10(4) MIRB-hNPCs. Fluorescence imaging, Perls' Prussian blue histochemistry, immunocytochemistry with SC121, a human-specific antibody, and T2-weighted magnetic resonance imaging ex vivo revealed there was increased homing and retention of MIRB-hNPCs in the injured cortex as compared to the control group in which MIRB-hNPCs were injected in the absence of a static magnetic field. Fluoro-Jade C staining and immunolabeling with specific markers confirmed the viability status of MIRB-hNPCs posttransplantation. These results show that increased homing and retention of MIRB-hNPCs post-TBI by applying a static magnetic field is a promising technique to deliver cells into the CNS for treatment of neurological injuries and neurodegenerative diseases.

Site-specific targeting of platelet-rich plasma via superparamagnetic nanoparticles.

BACKGROUND: Muscle strains are one of the most common injuries treated by physicians. Standard conservative therapy for acute muscle strains usually involves short-term rest, ice, and non-steroidal anti-inflammatory medications, but there is no clear consensus regarding treatments to accelerate recovery. Recently, clinical use of platelet-rich plasma (PRP) has gained momentum as an option for therapy and is appealing for many reasons, most notably because it provides growth factors in physiological proportions and it is autologous, safe, easily accessible, and potentially beneficial. Local delivery of patients' PRP to injured muscles can hasten recovery of function. However, specific targeting of PRP to sites of tissue damage in vivo is a major challenge that can limit its efficacy. HYPOTHESIS: Location of PRP delivery can be monitored and controlled in vivo with non-invasive tools. STUDY DESIGN: Controlled laboratory study using rats. METHODS: Superparamagnetic iron oxide nanoparticles (SPIONs) can be visualized by both MRI (in vivo) and fluorescence microscopy (after tissue harvesting). We labeled PRP with SPIONs and administered intramuscular injections of SPION-containing platelets. MRI was used to monitor the ability to manipulate and retain the location of PRP in vivo by placement of an external magnet. Platelets were isolated from whole blood and incubated with SPIONs. Following SPION incubation with PRP, a magnetic field was used to manipulate platelet location in culture dishes. In vivo, the tibialis anterior muscles (TAs) of anesthetized Sprague-Dawley rats were injected with SPION-containing platelets and MRI was used to track platelet position with and without a magnet worn over the TAs for 4 days. RESULTS: The method used to isolate PRP yielded a high concentration (almost 4-fold increase) of platelets. In vitro experiments show that the platelets successfully took up SPIONs and then rapidly responded to an applied magnetic field. Platelets without SPIONs did not respond to the magnetic field. In vivo experiments show that the SPION-containing platelets can be non-invasively maintained at a specific site with the application of a magnetic field. CONCLUSION: PRP may be a useful product in clinical treatment of muscle injuries, but one problem with using PRP as a therapeutic tool, is retaining PRP at the site of injury. We propose a potential solution with our findings that support this method at the cell, whole muscle, and in vivo levels. Controlling the location of PRP will allow the clustering of PRP to enrich the target area with growth factors and will prevent loss of the platelets over time at the site of injury.

Brain diabetic neurodegeneration segregates with low intrinsic aerobic capacity.

OBJECTIVES: Diabetes leads to cognitive impairment and is associated with age-related neurodegenerative diseases including Alzheimer's disease (AD). Thus, understanding diabetes-induced alterations in brain function is important for developing early interventions for neurodegeneration. Low-capacity runner (LCR) rats are obese and manifest metabolic risk factors resembling human "impaired glucose tolerance" or metabolic syndrome. We examined hippocampal function in aged LCR rats compared to their high-capacity runner (HCR) rat counterparts. METHODS: Hippocampal function was examined using proton magnetic resonance spectroscopy and imaging, unbiased stereology analysis, and a Y maze. Changes in the mitochondrial respiratory chain function and levels of hyperphosphorylated tau and mitochondrial transcriptional regulators were examined. RESULTS: The levels of glutamate, myo-inositol, taurine, and choline-containing compounds were significantly increased in the aged LCR rats. We observed a significant loss of hippocampal neurons and impaired cognitive function in aged LCR rats. Respiratory chain function and activity were significantly decreased in the aged LCR rats. Hyperphosphorylated tau was accumulated within mitochondria and peroxisome proliferator-activated receptor-gamma coactivator 1α, the NAD(+)-dependent protein deacetylase sirtuin 1, and mitochondrial transcription factor A were downregulated in the aged LCR rat hippocampus. INTERPRETATION: These data provide evidence of a neurodegenerative process in the hippocampus of aged LCR rats, consistent with those seen in aged-related dementing illnesses such as AD in humans. The metabolic and mitochondrial abnormalities observed in LCR rat hippocampus are similar to well-described mechanisms that lead to diabetic neuropathy and may provide an important link between cognitive and metabolic dysfunction.

Human neural progenitor cells retain viability, phenotype, proliferation, and lineage differentiation when labeled with a novel iron oxide nanoparticle, Molday ION Rhodamine B.

Ultrasmall superparamagnetic iron-oxide particles (USPIOs) loaded into stem cells have been suggested as a way to track stem cell transplantation with magnetic resonance imaging, but the labeling, and post-labeling proliferation, viability, differentiation, and retention of USPIOs within the stem cells have yet to be determined for each type of stem cell and for each type of USPIO. Molday ION Rhodamine B™ (BioPAL, Worcester, MA, USA) (MIRB) has been shown to be a USPIO labeling agent for mesenchymal stem cells, glial progenitor cells, and stem cell lines. In this study, we have evaluated MIRB labeling in human neuroprogenitor cells and found that human neuroprogenitor cells are effectively labeled with MIRB without use of transfection reagents. Viability, proliferation, and differentiation properties are unchanged between MIRB-labeled neuroprogenitors cells and unlabeled cells. Moreover, MIRB-labeled human neuroprogenitor cells can be frozen, thawed, and replated without loss of MIRB or even without loss of their intrinsic biology. Overall, those results show that MIRB has advantageous properties that can be used for cell-based therapy.

Defective thymic progenitor development and mature T-cell responses in a mouse model for Down syndrome.

In addition to archetypal cognitive defects, Down syndrome (DS) is characterized by altered lymphocyte development and function, including premature thymic involution and increased incidence of infections. However, the potential mechanisms for these changes have not been fully elucidated. The current study used the Ts65Dn mouse model of DS to assess deficiencies in T-cell development and possible molecular alterations. Ts65Dn mice exhibited premature thymic involution and a threefold to fourfold decrease in the number and proportion of immature, double-negative thymocyte progenitors. In addition, there were twofold fewer double-positive and CD4 single-positive thymocytes in Ts65Dn thymuses. Reflecting this deficient thymic function, there were fewer naive T cells in the spleen and polyclonal stimulation of peripheral T cells exhibited a marked reduction in proliferation, suggesting a senescent phenotype. In contrast, B-cell progenitors were unchanged in the bone marrow of Ts65Dn mice, but in the spleen, there were decreased transitional and follicular B cells and these cells proliferated less upon antigen receptor stimulus but not in response to lipopolysaccharide. As a potential mechanism for diminished thymic function, immature thymocyte populations expressed diminished levels of the cytokine receptor interleukin-7Rα, which was associated with decreased proliferation and increased apoptosis. Increased oxidative stress and inhibition of the Notch pathway were identified as possible mediators of decreased interleukin-7Rα expression in Ts65Dn mice. The data suggest that immature thymocyte defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signalling may alter lymphocyte development in Ts65Dn mice.

Coupling diffusion imaging with histological and gene expression analysis to examine the dynamics of cortical areas across the fetal period of human brain development.

As a prominent component of the human fetal brain, the structure of the cerebral wall is characterized by its laminar organization which includes the radial glial scaffold during fetal development. Diffusion tensor imaging (DTI) is useful to quantitatively delineate the microstructure of the developing brain and to clearly identify transient fetal layers in the cerebral wall. In our study, the spatio-temporal microstructural changes in the developing human fetal cerebral wall were quantitatively characterized with high-resolution DTI data of postmortem fetal brains from 13 to 21 gestational weeks. Eleven regions of interest for each layer in the entire cerebral wall were included. Distinctive time courses of microstructural changes were revealed for 11 regions of the neocortical plate. A histological analysis was also integrated to elucidate the relationship between DTI fractional anisotropy (FA) and histology. High FA values correlated with organized radial architecture in histological image. Expression levels of 17565 genes were quantified for each of 11 regions of human fetal neocortex from 13 to 21 gestational weeks to identify transcripts showing significant correlation with FA change. These correlations suggest that the heterogeneous and regionally specific microstructural changes of the human neocortex are related to different gene expression patterns.

Defective hematopoietic stem cell and lymphoid progenitor development in the Ts65Dn mouse model of Down syndrome: potential role of oxidative stress.

AIMS: Down Syndrome (DS), a genetic disease caused by a triplication of chromosome 21, is characterized by increased markers of oxidative stress. In addition to cognitive defects, patients with DS also display hematologic disorders and increased incidence of infections and leukemia. Using the Ts65Dn mouse model of DS, the goal of this study was to examine hematopoietic stem and lymphoid progenitor cell function in DS. RESULTS: Analysis of hematopoietic progenitor populations showed that Ts65Dn mice possessed fewer functional hematopoietic stem cells and a significantly decreased percentage of bone marrow lymphoid progenitors. Increased reactive oxygen species and markers of oxidative stress were detected in hematopoietic stem cell populations and were associated with a loss of quiescence. Bone marrow progenitor populations expressed diminished levels of the IL-7Rα chain, which was associated with decreased proliferation and increased apoptosis. Modulating oxidative stress in vitro suggested that oxidative stress selectively leads to decreased IL-7Rα expression, and inhibits the survival of IL-7Rα-expressing hematopoietic progenitors, potentially linking increased reactive oxygen species and immunopathology. INNOVATION: The study results identify a link between oxidative stress and diminished IL-7Rα expression and function. Further, the data suggest that this decrease in IL-7Rα is associated with defective hematopoietic development in Down Syndrome. CONCLUSION: The data suggest that hematopoietic stem and lymphoid progenitor cell defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signaling may alter hematologic development in Ts65Dn mice.

Sleep alterations in an environmental neurotoxin-induced model of parkinsonism.

Parkinson's disease (PD) is classically defined as a motor disorder resulting from decreased dopamine production in the basal ganglia circuit. In an attempt to better diagnose and treat PD before the onset of severe motor dysfunction, recent attention has focused on the early, non-motor symptoms, which include but are not limited to sleep disorders such as excessive daytime sleepiness (EDS) and REM behavioral disorder (RBD). However, few animal models have been able to replicate both the motor and non-motor symptoms of PD. Here, we present a progressive rat model of parkinsonism that displays disturbances in sleep/wake patterns. Epidemiological studies elucidated a link between the Guamanian variant of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) and the consumption of flour made from the washed seeds of the plant Cycas micronesica (cycad). Our study examined the effects of prolonged cycad consumption on sleep/wake activity in male, Sprague-Dawley rats. Cycad-fed rats exhibited an increase in length and/or number of bouts of rapid eye movement (REM) sleep and Non-REM (NREM) sleep at the expense of wakefulness during the active period when compared to control rats. This hypersomnolent behavior suggests an inability to maintain arousal. In addition, cycad-fed rats had significantly fewer orexin cells in the hypothalamus. Our results reveal a novel rodent model of parkinsonism that includes an EDS-like syndrome that may be associated with a dysregulation of orexin neurons. Further characterization of this early, non-motor symptom, may provide potential therapeutic interventions in the treatment of PD.

Environmental neurotoxin-induced progressive model of parkinsonism in rats.

OBJECTIVE: Exposure to a number of drugs, chemicals, or environmental factors can cause parkinsonism. Epidemiologic evidence supports a causal link between the consumption of flour made from the washed seeds of the plant Cycas micronesica by the Chamorro population of Guam and the development of amyotrophic lateral sclerosis/parkinsonism dementia complex. METHODS: We now report that consumption of washed cycad flour pellets by Sprague-Dawley male rats induces progressive parkinsonism. RESULTS: Cycad-fed rats displayed motor abnormalities after 2 to 3 months of feeding such as spontaneous unilateral rotation, shuffling gait, and stereotypy. Histological and biochemical examination of brains from cycad-fed rats revealed an initial decrease in the levels of dopamine and its metabolites in the striatum (STR), followed by neurodegeneration of dopaminergic (DAergic) cell bodies in the substantia nigra (SN) pars compacta (SNc). alpha-Synuclein (alpha-syn; proteinase K-resistant) and ubiquitin aggregates were found in the DAergic neurons of the SNc and neurites in the STR. In addition, we identified alpha-syn aggregates in neurons of the locus coeruleus and cingulate cortex. No loss of motor neurons in the spinal cord was found after chronic consumption of cycad flour. In an organotypic slice culture of the rat SN and the striatum, an organic extract of cycad causes a selective loss of dopamine neurons and alpha-syn aggregates in the SN. INTERPRETATION: Cycad-fed rats exhibit progressive behavioral, biochemical, and histological hallmarks of parkinsonism, coupled with a lack of fatality.

Locomotion analysis of Sprague-Dawley rats before and after injecting 6-OHDA.

The hypothesis that changes in measured ground reaction forces and time parameters during locomotion can noninvasively detect Parkinsonism in unilateral 6-OH dopamine (6-OHDA) lesioned rats is tested. It was found that changes of seven locomotion parameters can be used to construct a logistic regression model with a detection sensitivity and specificity of over 90% as compared to non-lesioned rats. Comparisons between this model and other neurological and neuromuscular disorders are presented.

Anatomical characterization of human fetal brain development with diffusion tensor magnetic resonance imaging.

The human brain is extraordinarily complex, and yet its origin is a simple tubular structure. Characterizing its anatomy at different stages of human fetal brain development not only aids in understanding this highly ordered process but also provides clues to detecting abnormalities caused by genetic or environmental factors. During the second trimester of human fetal development, neural structures in the brain undergo significant morphological changes. Diffusion tensor imaging (DTI), a novel method of magnetic resonance imaging, is capable of delineating anatomical components with high contrast and revealing structures at the microscopic level. In this study, high-resolution and high-signal-to-noise-ratio DTI data of fixed tissues of second-trimester human fetal brains were acquired and analyzed. DTI color maps and tractography revealed that important white matter tracts, such as the corpus callosum and uncinate and inferior longitudinal fasciculi, become apparent during this period. Three-dimensional reconstruction shows that major brain fissures appear while most of the cerebral surface remains smooth until the end of the second trimester. A dominant radial organization was identified at 15 gestational weeks, followed by both laminar and radial architectures in the cerebral wall throughout the remainder of the second trimester. Volumetric measurements of different structures indicate that the volumes of basal ganglia and ganglionic eminence increase along with that of the whole brain, while the ventricle size decreases in the later second trimester. The developing fetal brain DTI database presented can be used for education, as an anatomical research reference, and for data registration.

Measuring early pre-symptomatic changes in locomotion of SOD1-G93A rats--a rodent model of amyotrophic lateral sclerosis.

A locomotion analysis system for laboratory rats is presented. The system produces locomotion parameters (LPs) in 4 different domains: force, space, time and frequency. Video images of the walking rats are used to associate the system signals with individual limbs. Numerous LPs can be derived for every test run when the rat walks through the system on the way to sweets and a personal toy placed at the exit. This manuscript demonstrates that in order to differentiate SOD1-G93A mutant rat, a model of amyotrophic lateral sclerosis (ALS), from a Sprague Dawley (SD) control rat at a pre-symptomatic stage, one has only to use 8 key parameters. These 8 parameters are the bio-markers of ALS. The spline-based transformed values of these parameters are used as explanatory variables of a logistic regression model. This model predicts the probability that the examined rat belongs to the SOD1-G93A group. The model differentiates faultlessly between the SOD1 and control groups from the very first time the rats walked through the system at 51 days old. This system provides a new paradigm for ALS diagnosis, and it can have a significant impact on the development of new therapeutic procedures for ALS. The methodology presented in this manuscript can further address the development and validation of therapeutic procedures for other neurological diseases that affect locomotion.

Quantification of brain maturation and growth patterns in C57BL/6J mice via computational neuroanatomy of diffusion tensor images.

Diffusion Tensor magnetic resonance imaging and computational neuroanatomy are used to quantify postnatal developmental patterns of C57BL/6J mouse brain. Changes in neuronal organization and myelination occurring as the brain matures into adulthood are examined, and a normative baseline is developed, against which transgenic mice may be compared in genotype-phenotype studies. In early postnatal days, gray matter-based cortical and hippocampal structures exhibit high water diffusion anisotropy, presumably reflecting the radial neuronal organization. Anisotropy drops rapidly within a week, indicating that the underlying brain tissue becomes more isotropic in orientation, possibly due to formation of a complex randomly intertwined web of dendrites. Gradual white matter anisotropy increase implies progressively more organized axonal pathways, likely reflecting the myelination of axons forming tightly packed fiber bundles. In contrast to the spatially complex pattern of tissue maturation, volumetric growth is somewhat uniform, with the cortex and the cerebellum exhibiting slightly more pronounced growth. Temporally, structural growth rates demonstrate an initial rapid volumetric increase in most structures, gradually tapering off to a steady state by about 20 days. Fiber maturation reaches steady state in about 10 days for the cortex, to 30-40 days for the corpus callosum, the hippocampus, and the internal and external capsules.

Data mining in a behavioral test detects early symptoms in a model of amyotrophic lateral sclerosis.

"What's wrong with my genetically engineered animal?" is a common yet often difficult to answer question in behavioral phenotyping. We present here a method termed Pattern Array for mining movement patterns and isolating those that best capture an effect of a genetic manipulation. We demonstrate the method by searching for early motor symptoms in the open-field behavior of SOD1 mutant rats, an animal model of amyotrophic lateral sclerosis. Pattern Array was able to identify a unique motor pattern that differentiated the SOD1 mutants from the wild-type controls 2 months before disease onset. This pattern included heavy braking while moving near the arena wall but turning away from it. SOD1 mutants performed this pattern significantly less than wild-type controls in 2 independent data sets. At such early age the SOD1 mutants could not be differentiated from the controls by standard behavioral measures or by subjective observation. The early discovered symptom may enable investigators to test therapies aimed for intervention rather than remediation. Our results demonstrate the feasibility and potential of detecting subtle behavioral effects using data mining strategies.

Growth-compromised HSV-2 vector Delta RR protects from N-methyl-D-aspartate-induced neuronal degeneration through redundant activation of the MEK/ERK and PI3-K/Akt survival pathways, either one of which overrides apoptotic cascades.

We have previously shown that intrastriatal injection of Delta RR, the growth-compromised herpes simplex virus type 2 (HSV-2) vector for the antiapoptotic protein ICP10PK, prevents apoptosis caused by the excitotoxin N-methyl-D-aspartate (NMDA) in a mouse model of glutamatergic neuronal cell death (Golembewski et al. [2007] Exp. Neurol. 203:381-393). Because apoptosis regulation is stimulus and cell type specific, our studies were designed to examine the mechanism of Delta RR-mediated neuroprotection in striatal neurons. Organotypic striatal cultures (OSC) that retain much of the synaptic circuitry of the intact striatum were infected with Delta RR or a growth-compromised HSV-2 vector that lacks ICP10PK (Delta PK) and examined for neuroprotection-associated signaling. The mutated ICP10 proteins (p175 and p95) were expressed in 70-80% of neurons from Delta RR- and Delta PK-infected cultures, respectively, as determined by double-immunofluorescent staining with antibodies to ICP10 and NeuN or GAD65. Delta RR- but not Delta PK-treated OSC were protected from NMDA-induced apoptosis, as verified by ethidium homodimer staining, TUNEL, caspase-3 activation, and poly(AD-ribose) polymerase (PARP) cleavage. Neuroprotection was through ICP10PK-mediated activation of the survival pathways MEK/ERK and PI3-K/Akt, up-regulation of the antiapoptotic proteins Bag-1 and Bcl-2, and phosphorylation (inactivation) of the proapoptotic protein Bad. It was blocked by the MEK inhibitor U0126 or the PI3-K inhibitor LY294002, suggesting that either pathway can prevent NMDA-induced apoptosis. The data indicate that Delta RR-delivered ICP10PK stimulates redundant survival pathways that override proapoptotic cascades. Delta RR is a promising gene therapy platform against glutamatergic cell death.

The HSV-2 protein ICP10PK prevents neuronal apoptosis and loss of function in an in vivo model of neurodegeneration associated with glutamate excitotoxicity.

Excessive glutamate receptor activation results in neuronal death, a process known as excitotoxicity. Intrastriatal injection of N-methyl-d-aspartate (NMDA) is a model of excitotoxicity. We used this model to examine whether excitotoxic injury is inhibited by the anti-apoptotic herpes simplex virus type 2 (HSV-2) protein, ICP10PK, delivered by the replication incompetent HSV-2 vector, DeltaRR. Intrastriatal DeltaRR administration (2500 plaque forming units) was nontoxic and did not induce microglial activation 5 days after injection. Intrastriatal injection of DeltaRR with NMDA or 4 h after NMDA injection showed increased neuronal survival and decreased mitochondrial damage compared to injection of NMDA alone. Neuroprotection was due to the inhibition of NMDA-induced apoptosis through ERK activation. DeltaRR-treated mice did not develop NMDA-associated behavioral deficits. The data suggest that DeltaRR is a promising platform for treatment of acute neuronal injury.

White and gray matter development in human fetal, newborn and pediatric brains.

Brain anatomy is characterized by dramatic growth from the end of the second trimester through the neonatal stage. The characterization of normal axonal growth of the white matter tracts has not been well-documented to date and could provide important clues to understanding the extensive inhomogeneity of white matter injuries in cerebral palsy (CP) patients. However, anatomical studies of human brain development during this period are surprisingly scarce and histology-based atlases have become available only recently. Diffusion tensor magnetic resonance imaging (DTMRI) can reveal detailed anatomy of white matter. We acquired diffusion tensor images (DTI) of postmortem fetal brain samples and in vivo neonates and children. Neural structures were annotated in two-dimensional (2D) slices, segmented, measured, and reconstructed three-dimensionally (3D). The growth status of various white matter tracts was evaluated on cross-sections at 19-20 gestational weeks, and compared with 0-month-old neonates and 5- to 6-year-old children. Limbic, commissural, association, and projection white matter tracts and gray matter structures were illustrated in 3D and quantitatively characterized to assess their dynamic changes. The overall pattern of the time courses for the development of different white matter is that limbic fibers develop first and association fibers last and commissural and projection fibers are forming from anterior to posterior part of the brain. The resultant DTMRI-based 3D human brain data will be a valuable resource for human brain developmental study and will provide reference standards for diagnostic radiology of premature newborns.