Multimodal Evaluation of TMS - Induced Somatosensory Plasticity and Behavioral Recovery in Rats With Contusion Spinal Cord Injury

Introduction: Spinal cord injury (SCI) causes partial or complete damage to sensory and motor pathways and induces immediate changes in cortical function. Current rehabilitative strategies do not address this early alteration, therefore impacting the degree of neuroplasticity and subsequent recovery. The following study aims to test if a non-invasive brain stimulation technique such as repetitive transcranial magnetic stimulation (rTMS) is effective in promoting plasticity and rehabilitation, and can be used as an early intervention strategy in a rat model of SCI.Methods: A contusion SCI was induced at segment T9 in adult rats. An rTMS coil was positioned over the brain to deliver high frequency stimulation. Behavior, motor and sensory functions were tested in three groups: SCI rats that received high-frequency (20 Hz) rTMS within 10 min post-injury (acute-TMS; n = 7); SCI rats that received TMS starting 2 weeks post-injury (chronic-TMS; n = 5), and SCI rats that received sham TMS (no-TMS, n = 5). Locomotion was evaluated by the Basso, Beattie, and Bresnahan (BBB) and gridwalk tests. Motor evoked potentials (MEP) were recorded from the forepaw across all groups to measure integrity of motor pathways. Functional MRI (fMRI) responses to contralateral tactile hindlimb stimulation were measured in an 11.7T horizontal bore small-animal scanner.Results: The acute-TMS group demonstrated the fastest improvements in locomotor performance in both the BBB and gridwalk tests compared to chronic and no-TMS groups. MEP responses from forepaw showed significantly greater difference in the inter-peak latency between acute-TMS and no-TMS groups, suggesting increases in motor function. Finally, the acute-TMS group showed increased fMRI-evoked responses to hindlimb stimulation over the right and left hindlimb (LHL) primary somatosensory representations (S1), respectively; the chronic-TMS group showed moderate sensory responses in comparison, and the no-TMS group exhibited the lowest sensory responses to both hindlimbs.Conclusion: The results suggest that rTMS therapy beginning in the acute phase after SCI promotes neuroplasticity and is an effective rehabilitative approach in a rat model of SCI

Spinal cord organogenesis model reveals role of Flk1+ cells in self-organization of neural progenitor cells into complex spinal cord tissue

A platform for studying spinal cord organogenesis in vivo where embryonic stem cell (ESC)-derived neural progenitor cells (NPC) self-organize into spinal cord-like tissue after transplantation in subarachnoid space of the spinal cord has been described. We advance the applicability of this platform by imaging in vivo the formed graft through T2w magnetic resonance imaging (MRI). Furthermore, we used diffusion tensor imaging (DTI) to verify the stereotypical organization of the graft showing that it mimics the host spinal cord. Within the graft white matter (WM) we identified astrocytes that form glial limitans, myelinating oligodendrocytes, and myelinated axons with paranodes. Within the graft grey matter (GM) we identified cholinergic, glutamatergic, serotonergic and dopaminergic neurons. Furthermore, we demonstrate the presence of ESC-derived complex vasculature that includes the presence of blood brain barrier. In addition to the formation of mature spinal cord tissue, we describe factors that drive this process. Specifically, we identify Flk1+ cells as necessary for spinal cord formation, and synaptic connectivity with the host spinal cord and formation of host-graft chimeric vasculature as contributing factors. This model can be used to study spinal cord organogenesis, and as an in vivo drug discovery platform for screening potential therapeutic compounds and their toxicity. Keywords: Embryonic stem cells, Neural progenitor cells, Spinal cord organogenesis, Flk

Edx-17: A Novel, Safe and Efficacious Treatment for Sickle Cell Disease

Abstract Gene regulation of developmental hemoglobin switching holds the potential for therapeutic relief from all symptoms associated with Sickle Cell Disease (SCD). Reactivation of fetal gamma-globin expression (HbF) can replace mutant betaS-globin (HbS) to produce functional hemoglobin tetramers and eliminate the hemoglobin polymerization that is characteristic of sickled red blood cells. We have discovered a protein that regulates this developmental switch, and have identified a compound that stimulates expression of this protein. EdX-17 promotes expression of the anti-stress factor ferritin heavy chain (FtH), which enters the nucleus of erythroid precursor cells and activates expression of fetal gamma-globin, producing HbF (PNAS 98:9145-50, 2001; Blood 108:790a, 2006). Mononuclear cells were isolated from SCD patient blood and matured in vitro to the advanced erythroblast stage using a 28-day, 2-phase culture system (Methods in Molecular Biology 482:127-40, 2009; Blood 119:6296-306, 2012). Treatment with EdX-17 for 24h resulted in a dose-responsive induction of gamma-globin gene expression and a concomitant dose-responsive increase in HbF was observed after 28 days in culture. These studies demonstrate that EdX-17 doses in the picomolar range are sufficient to significantly enhance HbF. Furthermore, EdX-17 treatment reconstitutes fetal hemoglobin (HbF) in transgenic betaYAC mice to levels above 25-30% - the range thought to be sufficient to ameliorate symptoms of SCD – with no detectable ill effects. In fact, mice treated with EdX-17 tend to have shinier coats, are more alert and stronger than age-matched, untreated mice. Development of this novel therapeutic is expected to ameliorate SCD symptoms, decrease pain and morbidity, increase life-span, greatly improve patient quality of life, and significantly reduce treatment costs. Supported in part by The Sickle Cell Cure Foundation, Inc., the Bill & Melinda Gates Foundation, and EpimedX, LLC. Disclosures Broyles: EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Curtis:EpimedX, LLC: Employment, Equity Ownership, Research Funding. Roth:EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Floyd:EpimedX, LLC: Employment, Equity Ownership, Research Funding. Belegu:EpimedX, LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Floyd:EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding

Ferritin-H and a Phytotherapeutic, Alone or Combined, Reprogram RBC Precursor Cells From SCD Patients to Produce Levels of Fetal Hemoglobin That Constitute a Phenotypic Cure for Sickle Cell As Well As Providing Resistance to Malaria and a Probable Treatment for Beta-Thalassemia

Abstract Abstract 903 OBJECTIVE: To test the phenotypic cure we have discovered for SCD and malaria in vivo in mice and in vitro in human red blood cell precursors from SCD patients in two-phase cultures. BACKGROUND: Gene regulation of developmental hemoglobin switching provides phenotypic cures for beta thalassemias, sickle cell disease, and malaria since it is known that reactivation of fetal globin expression alleviates all these disorders. We discovered a protein that regulates this developmental switch. Ferritin heavy chain (FtH) represses adult β-globin and activates γ(fetal)-globin gene expression in embryonic/K562 erythroid cells (Broyles et al., PNAS98: 9145, 2001; US Patents #7,517,669, #7,718,669, & #2009/0232783 A1; EU Patent # EP1354032B1; Australia patent #2002217964), leading us to propose FtH as a therapeutic agent. METHODS: Normal C57BL/6 mice, transgenics carrying the complete human beta-globin gene cluster (Beta-YAC Tg), and transgenic mice that express human FtH in definitive erythroid cells have been used under an IACUC-approved protocol to show the safety and efficacy of human FtH and our phytotherapeutic in vivo. Erythroid precursor cells from pediatric SCD patients obtained under an IRB-approved protocol and cultured in a two-phase system allow direct testing of an FtH expression plasmid, FtH protein, and our phytotherapeutic for manipulating Hb phenotypes. Hemoglobins were identified and quantified by HPLC; fetal Hb was confirmed by immunofluorescence. RESULTS AND DISCUSSION: Human FtH transgenic mice, in which the FtH gene is driven by a truncated b-promoter lacking the FtH-binding motif, express human FtH in definitive erythroid cells which results in repression of bMajor-globin but not bMinor-globin. Thus, these TgFtH mice are born with a reduced ratio of bMajor/bMinor globins, resulting in α-globin chain excess and a mild b-thalassemia. In applying this therapy to humans, thalassemia would not be expected since FtH also activates g (fetal)-globin, preventing the chain imbalance. Recent results with normal mice and Beta-YacTg mice show that FtH protein and our phytotherapeutic are both well tolerated in vivo. We have used numbers of target cells (found in the FtH transgenics) as an initial screen for determining an effective dosing regimens. Initial results indicate that both therapeutics are altering the Hb phenotype as predicted. With cultured erythroid precursor cells from the pediatric SCD patients, our results show a complete switch from HbS to HbF with each mode of delivery of FtH or the phytotherapeutic. These results were replicated 15 times using cells from 9 SCD donors, as well as in erythroid precursors from normal donors. Human erythroid precursor cells have surface receptors specific for FtH. Immunofluorescence showed that human FtH is taken into the precursor cells in culture; and confocal microscopy confirmed nuclear localization of exogenously applied FtH. These results suggest that the purified protein and/or the phytotherapeutic can be directly delivered without gene therapy. Therefore, these methods of generating a phenotypic cure for malaria, beta-thal, and SCD should be inexpensive to deliver in vivo. Supported in part by donations to The Sickle Cell Cure Foundation and by a Grand Challenges in Global Health grant from the Bill & Melinda Gates Foundation. Disclosures: No relevant conflicts of interest to declare

RSNs with significant temporal structures.

<p>TOP Existence of significant (after correction for multiple comparisons) linear trends in three RSN outcome measures, namely the (a) spatial similarity (eta-squared, η²), (b) temporal signal fluctuation magnitude, and (c) BNC, are visualized using matrices. Red blocks indicate significant positive linear trend, blue blocks negative trend, and black boxes no significant trend. MIDDLE Existence of significant (after correction for multiple comparisons) annual periodicity in three RSN outcome measures. Red blocks indicate significant annual periodicity and black boxes no annual periodicity. BOTTOM AR orders of the estimated ARMA models for RSNs and RSN pairs are visualized for each outcome measures, where black box indicates no autocorrelation, red box AR order of 1, yellow box AR order of 2, and white box AR order of 3. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140134#pone.0140134.s006" target="_blank">S3 Table</a> for information on full ARMA model parameters.</p

Reproducibility of between-network connectivity (BNC) measurements.

<p>The combined BNC matrices show the degree of temporal synchrony between RSN pairs. Mean (a) and standard deviation (SD) (b) BNC values of the single- (below the main diagonal) and multi-participant (above the main diagonal) are shown. The diagonal elements were zeroed for display purposes. (c) Absolute value of the difference between the single- and the multi-participant BNC values. (d) Ten RSN pairs with the smallest (top) and the biggest (bottom) differences between single- and multi-participant mean BNC values. Mean BNC values from the single-subject dataset are overlaid as magenta circles on boxplots reporting on multi-participant data.</p

RSNs with significant linear trends in RSN outcome measures.

<p>Intercept and slope of the estimated linear trend, as well as the slope’s F statistic and p-value in three RSN outcome measures, namely the (a) spatial similarity (eta-squared, η²), (b) temporal fluctuation magnitude, and (c) BNC, for each RSNs with significant linear trends are listed.</p><p>RSNs with significant linear trends in RSN outcome measures.</p