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Neural progenitor cells (NPCs) derived from human pluripotent stem cells(hPSCs) provide major cell sources for repairing damaged neural circuitry and enabling axonal regeneration after spinal cord injury (SCI). However, the injury niche and inadequate intrinsic factors in the adult spinal cord restrict the therapeutic potential of transplanted NPCs. The Sonic Hedgehog protein (Shh) has crucial roles in neurodevelopment by promoting the formation of motorneurons and oligodendrocytes as well as its recently described neuroprotective features in response to the injury, indicating its essential role in neural homeostasis and tissue repair. In this study, we demonstrate that elevated SHH signaling in hNPCs by inhibiting its negative regulator, SUFU, enhanced cell survival and promoted robust neuronal differentiation with extensive axonal outgrowth, counteracting the harmful effects of the injured niche. Importantly, SUFU inhibition in NPCs exert non-cell autonomous effects on promoting survival and neurogenesis of endogenous cells and modulating the microenvironment by reducing suppressive barriers around lesion sites. The combined beneficial effects of SUFU inhibition in hNPCs resulted in the effective reconstruction of neuronal connectivity with the host and corticospinal regeneration, significantly improving neurobehavioral recovery in recipient animals. These results demonstrate that SUFU inhibition confers hNPCs with potent therapeutic potential to overcome extrinsic and intrinsic barriers in transplantation treatments for SCI., (© 2024. The Author(s).)

GABAergic interneurons are poised with the capacity to shape circuit output via inhibitory gating. How early in the development of medial vestibular nucleus (MVN) are GABAergic neurons recruited for feedforward shaping of outputs to higher centers for spatial navigation? The role of early GABAergic transmission in assembling vestibular circuits for spatial navigation was explored by neonatal perturbation. Immunohistochemistry and confocal imaging were utilized to reveal the expression of parvalbumin (PV)-expressing MVN neurons and their perineuronal nets. Whole-cell patch-clamp recording, coupled with optogenetics, was conducted in vitro to examine the synaptic function of MVN circuitry. Chemogenetic targeting strategy was also employed in vivo to manipulate neuronal activity during navigational tests. We found in rats a neonatal critical period before postnatal day (P) 8 in which competitive antagonization of GABAergic transmission in the MVN retarded maturation of inhibitory neurotransmission, as evidenced by deranged developmental trajectory for excitation/inhibition ratio and an extended period of critical period-like plasticity in GABAergic transmission. Despite increased number of PV-expressing GABAergic interneurons in the MVN, optogenetic-coupled patch-clamp recording indicated null-recruitment of these neurons in tuning outputs along the ascending vestibular pathway. Such perturbation not only offset output dynamics of ascending MVN output neurons, but was further accompanied by impaired vestibular-dependent navigation in adulthood. The same perturbations were however non-consequential when applied after P8. Results highlight neonatal GABAergic transmission as key to establishing feedforward output dynamics to higher brain centers for spatial cognition and navigation., (© 2024. The Author(s).)

Disruption of the blood-central nervous system barrier (BCB) is increasingly recognized as a pathological factor in diseases and trauma of the central nervous system. Despite the neuropathological impact, current treatment modalities do not target the BCB; strategies to reconstitute the impaired BCB have been restricted to nutritional and dietary remedies. As an integral cell type in the neurovascular unit, pericytes are crucial to the development, maintenance, and repair of the BCB. As such, pericytes are well poised as cellular agents for reconstitution of the impaired BCB. Here, we summarize recent revelations regarding the role of BCB disruption in diseases and trauma of the central nervous system and highlight how pericytes are harnessed to provide targeted therapeutic effect in each case. This review will also address how recent advances in pericyte derivation strategies can serve to overcome practical hurdles in the clinical use of pericytes., (© 2024 The Authors.)

Neural stem cells (NSCs) derived from human pluripotent stem cells (hPSCs) are considered a major cell source for reconstructing damaged neural circuitry and enabling axonal regeneration. However, the microenvironment at the site of spinal cord injury (SCI) and inadequate intrinsic factors limit the therapeutic potential of transplanted NSCs. Here, it is shown that half dose of SOX9 in hPSCs-derived NSCs (hNSCs) results in robust neuronal differentiation bias toward motor neuron lineage. The enhanced neurogenic potency is partly attributed to the reduction of glycolysis. These neurogenic and metabolic properties retain after transplantation of hNSCs with reduced SOX9 expression in a contusive SCI rat model without the need for growth factor-enriched matrices. Importantly, the grafts exhibit excellent integration properties, predominantly differentiate into motor neurons, reduce glial scar matrix accumulation to facilitate long-distance axon growth and neuronal connectivity with the host as well as dramatically improve locomotor and somatosensory function in recipient animals. These results demonstrate that hNSCs with half SOX9 gene dosage can overcome extrinsic and intrinsic barriers, representing a powerful therapeutic potential for transplantation treatments for SCI., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The in vitro derivation of Schwann cells from human bone marrow stromal cells (hBMSCs) opens avenues for autologous transplantation to achieve remyelination therapy for post-traumatic neural regeneration. Towards this end, we exploited human induced pluripotent stem-cell-derived sensory neurons to direct Schwann-cell-like cells derived from among the hBMSC-neurosphere cells into lineage-committed Schwann cells (hBMSC-dSCs). These cells were seeded into synthetic conduits for bridging critical gaps in a rat model of sciatic nerve injury. With improvement in gait by 12-week post-bridging, evoked signals were also detectable across the bridged nerve. Confocal microscopy revealed axially aligned axons in association with MBP-positive myelin layers across the bridge in contrast to null in non-seeded controls. Myelinating hBMSC-dSCs within the conduit were positive for both MBP and human nucleus marker HuN. We then implanted hBMSC-dSCs into the contused thoracic cord of rats. By 12-week post-implantation, significant improvement in hindlimb motor function was detectable if chondroitinase ABC was co-delivered to the injured site; such cord segments showed axons myelinated by hBMSC-dSCs. Results support translation into a protocol by which lineage-committed hBMSC-dSCs become available for motor function recovery after traumatic injury to both peripheral and central nervous systems.

Vestibular information processed first by the brainstem vestibular nucleus (VN), and further by cerebellum and thalamus, underlies diverse brain function. These include the righting reflexes and spatial cognitive behaviour. While the cerebellar and thalamic circuits that decode vestibular information are known, the importance of VN neurons and the temporal requirements for their maturation that allow developmental consolidation of the aforementioned circuits remains unclear. We show that timely unsilencing of glutamatergic circuits in the VN by NMDA receptor-mediated insertion of AMPAR receptor type 1 (GluA1) subunits is critical for maturation of VN and successful consolidation of higher circuits that process vestibular information. Delayed unsilencing of NMDA receptor-only synapses of neonatal VN neurons permanently decreased their functional connectivity with inferior olive circuits. This was accompanied by delayed pruning of the inferior olive inputs to Purkinje cells and permanent reduction in their plasticity. These derangements led to deficits in associated vestibular righting reflexes and motor co-ordination during voluntary movement. Vestibular-dependent recruitment of thalamic neurons was similarly reduced, resulting in permanently decreased efficiency of spatial navigation. The findings thus show that well-choreographed maturation of the nascent vestibular circuitry is prerequisite for functional integration of vestibular signals into ascending pathways for diverse vestibular-related behaviours., Competing Interests: Conflict of interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Transplantation of oligodendrocyte precursors (OPs) is potentially therapeutic for myelin disorders but a safe and accessible cell source remains to be identified. Here we report a two-step protocol for derivation of highly enriched populations of OPs from bone marrow stromal cells of young adult rats (aMSCs). Neural progenitors among the aMSCs were expanded in non-adherent sphere-forming cultures and subsequently directed along the OP lineage with the use of glial-inducing growth factors. Immunocytochemical and flow cytometric analyses of these cells confirmed OP-like expression of Olig2, PDGFRα, NG2, and Sox10. OPs so derived formed compact myelin both in vitro, as in co-culture with purified neurons, and in vivo, following transplantation into the corpus callosum of neonatal shiverer mice. Not only did the density of myelinated axons in the corpus callosum of recipient shiverer mice reach levels comparable to those in age-matched wild-type mice, but the mean lifespan of recipient shiverer mice also far exceeded those of non-recipient shiverer mice. Our results thus promise progress in harnessing the OP-generating potential of aMSCs towards cell therapy for myelin disorders.

Key Points: Chemogenetic activation of medial vestibular nucleus-projecting 5-HT neurons resulted in deficits in vestibular-mediated tasks, including negative geotaxis, balance beam and rota-rod tests. The 5-HT 1A receptor mediates the vestibular-related behavioural effects of 5-HT in the vestibular nucleus. 5-HT 1A receptor activation attenuated evoked excitatory postsynaptic currents and evoked inhibitory postsynaptic currents via a presynaptic mechanism in the vestibular nucleus., Abstract: While the anxiolytic effects of serotonergic neuromodulation are well studied, its role in sensorimotor coordination and postural control is unclear. In this study, we show that an increase of serotonin (5-hydroxytryptamine, 5-HT) at the medial vestibular nucleus (MVN), a brainstem centre for vestibulospinal coordination, by either direct cannula administration or chemogenetic stimulation of MVN-projecting serotonergic neurons, adversely affected performance of rats in vestibular-mediated tasks, including negative geotaxis, balance beam and rota-rod tests. Application of the 5-HT 1 and 5-HT 7 receptor co-agonist 8-hydroxy-2-(di-n-propylamino) tetralin recapitulated the effect of 5-HT, while co-administration of the specific 5-HT 1A receptor antagonist WAY 100135 effectively abolished all 5-HT-induced behavioural deficits. This indicated that 5-HT 1A receptors mediated the effects of 5-HT in the rat MVN. Using whole-cell patch-clamp recording, we demonstrated that 5-HT 1A receptor activation attenuated both evoked excitatory and evoked inhibitory postsynaptic currents through a presynaptic mechanism in the rat MVN. The results thus highlight the 5-HT 1A receptor as the gain controller of vestibular-related brainstem circuits for posture and balance., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

The phenotypic instability of adult tissue-derived Schwann cell-like cells (SCLCs) as revealed upon withdrawal of glia-inducing culture supplements limits their clinical utility for cell therapy and disease modelling. We previously overcame this limitation by co-culturing bone marrow-derived SCLCs with neurons purified from developing rat and subsequently human sensory neurons such that direct contact between cell types accomplished the cell-intrinsic switch to the Schwann cell fate. Here, our search for juxtacrine instructive signals found both Notch ligands and neuregulin-1 type III localized on the surface of DRG neurons via live cell immunocytochemistry. Bypassing ligand-induced release of the Notch intracellular domain (NICD) by transient transfection of SCLCs with the pAdlox/V5-His-NICD construct was shown to upregulate ErbB2/3. Interaction of ErbB2/3 with neuregulin-1 type III (NRG1 type III) as presented on neurons then mediated the switch to the Schwann cell fate as demonstrated by expression of S100β/p75/ Sox10/Krox20. In contrast, treatment of cocultures with γ-secretase inhibitor perturbed Notch signalling in SCLCs and consequently deterred both upregulation of ErbB2/3 and the transition to the Schwann cell fate. Taken together, juxtacrine signalling via Notch is key to the upregulation of ErbB receptors for neuregulin-driven commitment of SCLCs to the Schwann cell fate., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK -/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory., Competing Interests: The authors declare no conflict of interest.

Perineuronal nets (PN) restrict neuronal plasticity in the adult brain. We hypothesize that activity-dependent consolidation of PN is required for functional maturation of behavioral circuits. Using the postnatal maturation of brainstem vestibular nucleus (VN) circuits as a model system, we report a neonatal period in which consolidation of central vestibular circuitry for graviception is accompanied by activity-dependent consolidation of chondroitin sulfate (CS)-rich PN around GABAergic neurons in the VN. Postnatal onset of negative geotaxis was used as an indicator for functional maturation of vestibular circuits. Rats display negative geotaxis from postnatal day (P) 9, coinciding with the condensation of CS-rich PN around GABAergic interneurons in the VN. Delaying PN formation, by removal of primordial CS moieties on VN with chondroitinase ABC (ChABC) treatment at P6, postponed emergence of negative geotaxis to P13. Similar postponement was observed following inhibition of GABAergic transmission with bicuculline, in line with the reported role of PN in increasing excitability of parvalbumin neurons. We further reasoned that PN-CS restricts bioavailability of plasticity-inducing factors such as semaphorin 3A (Sema3A) to bring about circuit maturation. Treatment of VN explants with ChABC to liberate PN-bound Sema3A resulted in dendritic growth and arborization, implicating structural plasticity that delays synapse formation. Evidence is thus provided for the role of PN-CS-Sema3A in regulating structural and circuit plasticity at VN interneurons with impacts on the development of graviceptive postural control.

Schwann cell-seeded nerve guidance channels are designed to assist post-traumatic nerve regeneration in the PNS. Chitosan is a natural polymer well suited for tissue engineering as it is biocompatible, non-immunogenic, and biodegradable. Electrospun chitosan nanofibers utilized in nerve guidance channels have the capacity for guiding axonal growth within the channel lumen yet are limited in their capacity to maintain structural integrity within physiological environments. To address this, we attempted genipin crosslinking of chitosan nanofibers. Compared to neat chitosan nanofibers, genipin-treated nanofibers exhibited increased stiffness, resistance to swelling and lysozymal degradation. Furthermore, alignment and proliferation of purified Schwann cell cultures upon genipin-treated substratum was enhanced. When dorsal root ganglion explants were utilized as an in vitro model of peripheral nerve regeneration, emigrating neurons and Schwann cells assumed the uniaxial pattern of aligned electrospun chitosan nanofibers. Neurite growth along the nanofibers led, reaching a frontier more than twice that of the pursuant Schwann cells. Critically, neurite growth rate upon genipin-treated nanofibers demonstrated a 100% increase. Altogether, genipin treatment improves upon the physical and biological properties of chitosan nanofibers towards their utility in nerve guidance channel design., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Our ultimate goal of in vitro derivation of Schwann cells (SCs) from adult bone marrow stromal cells (BMSCs) is such that they may be used autologously to assist post-traumatic nerve regeneration. Existing protocols for derivation of SC-like cells from BMSCs fall short in the stability of the acquired phenotype and the functional capacity to myelinate axons. Our experiments indicated that neuro-ectodermal progenitor cells among the human hBMSCs could be selectively expanded and then induced to differentiate into SC-like cells. Co-culture of the SC-like cells with embryonic dorsal root ganglion neurons facilitated contact-mediated signaling that accomplished the switch to fate-committed SCs. Microarray analysis and in vitro myelination provided evidence that the human BMSC-derived SCs were functionally mature. This was reinforced by repair and myelination phenotypes observable in vivo with the derived SCs seeded into a nerve guide as an implant across a critical gap in a rat model of sciatic nerve injury., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing β-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100β/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and growth factors to bias cell-signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. In this study, we took an alternative approach and selected 5 small-molecule inhibitors of key signaling pathways in an 8-day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium resulted in neuronal networks immunopositive for synaptic vesicle markers and vesicular glutamate transporters suggestive of excitatory neurotransmission. Subpopulations of the derived neurons were electrically excitable, showing tetrodotoxin-sensitive action potentials in patch-clamp experiments. Coculture of the derived neurons with rat Schwann cells under myelinating conditions resulted in upregulated levels of neuronal neuregulin 1 type III in conjunction with the phosphorylated receptors ErbB2 and ErbB3, consistent with amenability of the neuritic network to myelination. As surrogates of embryonic dorsal root ganglia neurons, the derived sensory neurons provided contact-dependent cues to commit bone marrow-derived Schwann cell-like cells to the Schwann cell fate. Our rapid and efficient induction protocol promises not only controlled differentiation of human iPSCs into sensory neurons, but also utility in the translation to a protocol whereby human bone marrow-derived Schwann cells become available for autologous transplantation and remyelination therapy. Stem Cells Translational Medicine 2017;6:369-381., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Background: Bone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for ex vivo generation of neurons and glia. Limited numbers of this subpopulation, however, hinder translation into autologous cell-based therapy. Here, we demonstrate rapid and efficient conditioning with hypoxia to enrich for these neural progenitor cells prior to further expansion in neurosphere culture., Method: Adherent cultures of BMSCs (rat/human) were subjected to 1 % oxygen for 24 h and then subcultured as neurospheres with epidermal growth factor (EGF) and basic fibroblast growth factor supplementation. Neurospheres and cell progeny were monitored immunocytochemically for marker expression. To generate Schwann cell-like cells, neurospheres were plated out and exposed to gliogenic medium. The resulting cells were co-cultured with purified dorsal root ganglia (rat) neurons and then tested for commitment to the Schwann cell fate. Fate-committed Schwann cells were subjected to in vitro myelination assay., Results: Transient hypoxic treatment increased the size and number of neurospheres generated from both rat and human BMSCs. This effect was EGF-dependent and attenuated with the EGF receptor inhibitor erlotinib. Hypoxia did not affect the capacity of neurospheres to generate neuron- or glia-like precursors. Human Schwann cell-like cells generated from hypoxia-treated BMSCs demonstrated expression of S100β /p75 and capacity for myelination in vitro., Conclusion: Enhancing the yield of neural progenitor cells with hypoxic preconditioning of BMSCs in vitro but without inherent risks of genetic manipulation provides a platform for upscaling production of neural cell derivatives for clinical application in cell-based therapy.

The recognition of head orientation in the adult involves multi-level integration of inputs within the central vestibular circuitry. How the different inputs are recruited during postnatal development remains unclear. We hypothesize that glutamatergic transmission at the vestibular nucleus contributes to developmental registration of head orientations along the vestibulo-olivary pathway. To investigate the maturation profile by which head rotational signals are registered in the brainstem, we used sinusoidal rotations on the orthogonal planes of the three pairs of semicircular canals. Fos expression was used as readout of neurons responsive to the rotational stimulus. Neurons in the vestibular nucleus and prepositus hypoglossal nucleus responded to all rotations as early as P4 and reached adult numbers by P21. In the reticular formation and inferior olive, neurons also responded to horizontal rotations as early as P4 but to vertical rotations not until P21 and P25, respectively. Neuronal subpopulations that distinguish between rotations activating the orthogonally oriented vertical canals were identifiable in the medial and spinal vestibular nuclei by P14 and in the inferior olivary subnuclei IOβ and IOK by P25. Neonatal perturbation of glutamate transmission in the vestibular nucleus was sufficient to derange formation of this distribution in the inferior olive. This is the first demonstration that developmental refinement of glutamatergic synapses in the central vestibular circuitry is essential for developmental registration of head rotational signals in the brainstem.

Cigarette smoke induces injury and neutrophilic inflammation in the airways of smokers. The stability and activity of inflammatory effectors, IL8 and neutrophil elastase (NE), can be prolonged by binding to airway heparan sulfate (HS)/syndecan-1, posing risk for developing chronic obstructive pulmonary disease(COPD). We hypothesize that antagonizing HS/syndecan-1 binding of the inflammatory effectors could reduce smoking-related neutrophil-mediated airway inflammation. Analysis of bronchoalveolar lavage fluid(BALF) of COPD patients found both total and unopposed NE levels to be significantly higher among smokers with COPD than non-COPD subjects. Similar NE burden was observed in smoke-exposed rats compared to sham air controls. We chose sulfated-maltoheptaose(SM), a heparin-mimetic, to antagonize HS/sydecan-1 binding of the inflammatory mediators in airway fluids and lung tissues of the smoke-exposed rat model. Airway treatment with SM resulted in displacement of CINC-1 and NE from complexation with bronchio-epithelial HS/syndecan-1, dissipating the chemokine gradient for neutrophil flux across to the bronchial lumen. Following SM displacement of NE from shed HS/syndecan-1 in bronchial fluids, NE became accessible to inhibition by α1-antitrypsin endogenous in test samples. The antagonistic actions of SM against syndecan-1 binding of NE and CINC-1 in smoke-exposed airways suggest new therapeutic opportunities for modulating airway inflammation in smokers with SM delivery.

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene-gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21-NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The capability of the central vestibular system in utilizing cues arising from the inner ear determines the ability of animals to acquire the sense of head orientations in the three-dimensional space and to shape postural movements. During development, neurons in the vestibular nucleus (VN) show significant changes in their electrophysiological properties. An age-dependent enhancement of membrane excitability is accompanied by a progressive increase in firing rate and discharge regularity. The coding of horizontal and vertical linear motions also exhibits developmental refinement in VN neurons. Further, modification of cell surface receptors, such as glutamate receptors, of developing VN neurons are well-orchestrated in the course of maturation, thereby regulating synaptic efficacy and spatial coding capacity of these neurons in local circuits. Taken together, these characteristic features of VN neurons contribute to developmental establishment of space-centered coordinates within the brain.

The availability of human stem cells heralds a new era for in vitro cell-based modeling of neurodevelopmental and neurodegenerative diseases. Adding to the excitement is the discovery that somatic cells of patients can be reprogrammed to a pluripotent state from which neural lineage cells that carry the disease genotype can be derived. These in vitro cell-based models of neurological diseases hold promise for monitoring of disease initiation and progression, and for testing of new drug treatments on the patient-derived cells. In this review, we focus on the prospective applications of different stem cell types for disease modeling and drug screening. We also highlight how the availability of patient-specific induced pluripotent stem cells (iPS cells) offers a unique opportunity for studying and modeling human neurodevelopmental and neurodegenerative diseases in vitro and for testing small molecules or other potential therapies for these disorders. Finally, the limitations of this technology from the standpoint of reprogramming efficiency and therapeutic safety are discussed.

It stems from the magnetism: The extraction of stem/progenitor cells from the brain of live animals is possible using antibodies conjugated to magnetic nanoparticles (Ab-MNPs). The Ab-MNPs are introduced to a rat's brain with a superfine micro-syringe. The stem cells attach to the Ab-MNPs and are magnetically isolated and removed. They can develop into neurospheres and differentiate into different types of cells outside the subject body. The rat remains alive and healthy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using sinusoidal oscillations of linear acceleration along both the horizontal and vertical planes to stimulate otolith organs in the inner ear, we charted the postnatal time at which responsive neurons in the rat inferior olive (IO) first showed Fos expression, an indicator of neuronal recruitment into the otolith circuit. Neurons in subnucleus dorsomedial cell column (DMCC) were activated by vertical stimulation as early as P9 and by horizontal (interaural) stimulation as early as P11. By P13, neurons in the β subnucleus of IO (IOβ) became responsive to horizontal stimulation along the interaural and antero-posterior directions. By P21, neurons in the rostral IOβ became also responsive to vertical stimulation, but those in the caudal IOβ remained responsive only to horizontal stimulation. Nearly all functionally activated neurons in DMCC and IOβ were immunopositive for the NR1 subunit of the NMDA receptor and the GluR2/3 subunit of the AMPA receptor. In situ hybridization studies further indicated abundant mRNA signals of the glutamate receptor subunits by the end of the second postnatal week. This is reinforced by whole-cell patch-clamp data in which glutamate receptor-mediated miniature excitatory postsynaptic currents of rostral IOβ neurons showed postnatal increase in amplitude, reaching the adult level by P14. Further, these neurons exhibited subthreshold oscillations in membrane potential as from P14. Taken together, our results support that ionotropic glutamate receptors in the IO enable postnatal coding of gravity-related information and that the rostral IOβ is the only IO subnucleus that encodes spatial orientations in 3-D.

Vestibular information arising from rotational head movement and that from translational head movement are detected respectively by the semicircular canal and otolith organ in the inner ear. Spatiotemporal cues are in turn processed by the vestibulo-olivo-cerebellar pathway for sensorimotor coordination, but the role of the inferior olive (IO) in this pathway remains unclear. To address whether rotational and translational movements are differentially represented in the IO, we studied the distribution pattern of IO neurons recruited into the circuitry following selective activation of receptor hair cells of the horizontal semicircular canal or the utricle in adult rats. Neurons in the beta nucleus of IO (IOβ) and dorso-medial cell column of IO were responsive to horizontal translation, but not rotation. Notably, otolith-related neurons were observable largely in the rostral IOβ. In contrast, the subnucleus A of IO (IOA), subnucleus C of IO (IOC), and dorsal cap of Kooy (IOK) were responsive to horizontal rotation, but not translation. In the IOA, these canal-related neurons were clustered in the medial portion of the subnucleus. In the IOC, canal-related neurons were skewed towards the rostral half. In the IOK, canal-related neurons were found throughout the subnucleus. These indicate that the distributions of canal- and otolith-related neurons encoding horizontal motions are clearly segregated in the IO. These discrete IO subnuclei therefore provide a topographic map for temporal and adaptive operations of sensorimotor coordination and spatial reference.

We examined the maturation expression profile of tyrosine kinase B (TrkB) receptor in rat vestibular nuclear neurons that were activated by sinusoidal linear acceleration along the horizontal or vertical axis. The otolithic origin of Fos expression in these neurons was confirmed with labyrinthectomized controls and normal controls, which showed only sporadically scattered Fos-labeled neurons in the vestibular nucleus. In P4-6 test rats, no Fos-labeled neurons were found in the vestibular nucleus, but the medial and spinal vestibular neurons showed weak immunoreactivity for TrkB. The intensity of TrkB immunoreactivity in vestibular nuclear neurons progressively increased in the second postnatal week but remained low in adults. From P7 onward, TrkB-expressing neurons responded to horizontal or vertical otolithic stimulation with Fos expression. The number of Fos-labeled vestibular nuclear neurons expressing TrkB increased with age, from 13-43% in P7 rats to 85-90% in adult rats. Our results therefore suggest that TrkB/neurotrophin signaling plays a dominant role in modulating vestibular nuclear neurons for the coding of gravity-related horizontal head movements and for the regulation of vestibular-related behavior during postnatal development., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Establishing correct neuronal circuitry is crucial to proper function of the vertebrate nervous system. The abundance of chondroitin sulfate (CS) proteoglycans in embryonic neural environments suggests that matrix proteoglycans regulate axonal projections when fiber tracts have not yet formed. Among the early-born neurons, the vestibular nucleus (VN) neurons initiate commissural projections soon after generation at E12.5 and reach the contralateral target by E15.5 in the rat hindbrain. We therefore exploited 24-hour cultures (1 day in vitro (DIV)) of the rat embryos and chondroitinase ABC treatment of the hindbrain matrix to reveal the role of CS moieties in axonal initiation and projection in the early hindbrain., Results: DiI tracing from the VN at E12.5(+1 DIV) showed contralaterally projecting fibers assuming fascicles that hardly reached the midline in the controls. In the enzyme-treated embryos, the majority of fibers were unfasciculated as they crossed the midline at 90°. At E13.5(+1 DIV), the commissural projections formed fascicles and crossed the midline in the controls. Enzyme treatment apparently did not affect the pioneer axons that had advanced as thick fascicles normal to the midline and beyond, towards the contralateral VN. Later projections, however, traversed the enzyme-treated matrix as unfasciculated fibers, deviated from the normal course crossing the midline at various angles and extending beyond the contralateral VN. This suggests that CSs also limit the course of the later projections, which otherwise would be attracted to alternative targets., Conclusions: CS moieties in the early hindbrain therefore control the course and fasciculation of axonal projections and the timing of axonal arrival at the target.

Schwann cells are critically important for tissue repair, axonal regrowth and remyelination following injury to peripheral nerves. The absence of Schwann cells or an equivalent cell type in the central nervous system (CNS) may limit the regeneration capacity of the CNS. Mesenchymal stem cells (MSCs) have therefore been investigated for their potential to be induced to develop a Schwann cell phenotype. The methods for derivation of Schwann cell-like cells from MSCs and the benefits and limitations of each of these methods are presented in this review. Issues related to instability of the derived Schwann cell phenotype, apoptosis of derived cells in transplants, and the inability to predict with confidence how the cells will behave after transplantation are discussed. Finally, we suggest the need for further elucidation of the biology of Schwann cell differentiation and the signals for their derivation from MSC, in order to resolve these obstacles and to enable transplantation of MSC-derived Schwann cells as a therapeutic strategy in CNS injury.

Mesenchymal stem cell (MSC)-based engineering is promising for cartilage repair. However, the compositional mechanical relationship of the engineered structures has not been extensively studied, given the importance of such relationship in native cartilage tissues. In this study, a novel human MSC-collagen microsphere system was used to study the compositional mechanical relationship during in vitro chondrogenic differentiation using histological and biochemical methods and a microplate compression assay. The mechanical property was found positively correlating with newly deposited cartilage-relevant matrices, glycosaminoglycan, and type II collagen, and with the collagen crosslinker density, in agreement with the presence of thick collagen bundles upon structural characterization. On the other hand, the mechanical property negatively correlates with type I collagen and total collagen, suggesting that the initial collagen matrix scaffold of the microsphere system was being remodeled by the differentiating human MSCs. This study also demonstrated the application of a simple, sensitive, and nondestructive tool for monitoring the progression of chondrogenic differentiation of MSCs in tissue-engineered constructs and therefore contributes to future development of novel cartilage repair strategies.

Glutamate transmission from vestibular end organs to central vestibular nuclear complex (VNC) plays important role in transferring sensory information about head position and movements. Three isoforms of vesicular glutamate transporters (VGLUTs) have been considered so far the most specific markers for glutamatergic neurons/cells. In this study, VGLUT1 and VGLUT2 were immunohistochemically localized to axon terminals in VNC and somata of vestibular primary afferents in association with their central and peripheral axon endings, and VGLUT1 and VGLUT3 were co-localized to hair cells of otolith maculae and cristae ampullaris. VGLUT1 and VGLUT2 defined three subsets of Scarpa's neurons (vestibular ganglionic neurons): those co-expressing VGLUT1 and VGLUT2 or expressing only VGLUT2, and those expressing neither. In addition, many neurons located in all vestibular subnuclei were observed to contain hybridized signals for VGLUT2 mRNA and a few VNC neurons, mostly scattered in medial vestibular nucleus (MVe), displayed VGLUT1 mRNA labelling. Following unilateral ganglionectomy, asymmetries of VGLUT1-immunoreactivity (ir) and VGLUT2-ir occurred between two VNCs, indicating that the VNC terminals containing VGLUT1 and/or VGLUT2 are partly of peripheral origin. The present data indicate that the constituent cells/neurons along the vestibular pathway selectively apply VGLUT isoforms to transport glutamate into synaptic vesicles for glutamate transmission., (© 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Autologous nerve grafts have been the 'gold standard' for treatment of peripheral nerve defects that exceed the critical gap length. To address issues of limited availability of donor nerves and donor site morbidity, we have fabricated chitosan conduits and seeded them with bone marrow stromal cell (BMSC)-derived Schwann cells as an alternative. The derived Schwann cells used were checked for fate commitment. The conduits were tested for efficacy in bridging the critical gap length of 12 mm in sciatic nerves of adult rats. By three months post-operation, mid-shank circumference, nerve conduction velocity, average regenerated myelin area, and myelinated axon count, in nerves bridged with BMSC-derived Schwann cells were similar to those treated with sciatic nerve-derived Schwann cells (p > 0.05) but significantly higher than those bridged with PBS-filled conduits (p < 0.05). Evidence is thus provided in support of the use of chitosan conduits seeded with BMSC-derived Schwann cells to treat critical defects in peripheral nerves. This provides the basis to pursue BMSC as an autologous source of Schwann cells for transplantation therapy in larger animal species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Multiple osteochondromas (MO) is an autosomal-dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice-site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173 + 1G > T (EXT2) mutation was identified in a three-generation 34-member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon-reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation., (© 2010 Orthopaedic Research Society.)

Background: Crystallization is believed to be the initiation step of urolithiasis, even though it is unknown where inside the nephron the first crystal nucleation occurs., Methods: Direct nucleation of calcium oxalate and subsequent events including crystal retention, cellular damage, endocytosis, and hyaluronan (HA) expression, were tested in a two-compartment culture system with intact human proximal tubular HK-2 cell monolayer., Results: Calcium oxalate dihydrate (COD) was nucleated and bound onto the apical surface of the HK-2 cells under hypercalciuric and hyperoxaluric conditions. These cells displayed mild cellular damage and internalized some of the adhered crystals within 18h post-COD-exposure, as revealed by electron microscopy. Prolonged incubation in complete medium caused significant damage to disrupt the monolayer integrity. Furthermore, hyaluronan disaccharides were detected in the harvested media, and were associated with HAS-3 mRNA expression., Conclusion: Human proximal cells were able to internalize COD crystals which nucleated directly onto the apical surface, subsequently triggering cellular damage and HAS-3 specific hyaluronan synthesis as an inflammatory response. The proximal tubule cells here demonstrate that it plays an important role in facilitating urolithiasis via endocytosis and creating an inflammatory environment whereby free hyaluronan in tubular fluid can act as crystal-binding molecule at the later segments of distal and collecting tubules., (Copyright 2010 Elsevier B.V. All rights reserved.)

Schwann cell transplantation improves post-traumatic nerve regeneration in both PNS and CNS but sufficient numbers of immunocompatible cells are required for clinical application. Currently, Schwann cell-like cells derived from the bone marrow lack fate commitment and revert to a fibroblast-like phenotype upon withdrawal of differentiation-inducing factors. In recapitulation of embryonic events leading to Schwann cell maturation, we hypothesize that the Schwann cell-like cells acquire the switch to fate commitment through contact-dependent cues from incipient neurons of the developing dorsal root ganglia. To address this, Schwann cell-like cells derived from adult rat bone marrow were cocultured with neurons purified from embryonic dorsal root ganglia. A cell-intrinsic switch to the Schwann cell fate was achieved consistently and the cell progeny maintained expression of the markers S100 beta, p75(NTR) , GFAP, P0 and Sox 10 even without exogenous differentiation-inducing factors or neurons. In vitro formation of MBP-positive segments under myelinating conditions by the cell progeny was comparable to that by sciatic nerve-derived Schwann cells. Controls in which Schwann cell-like cells were barred from direct contact with neurons in coculture reverted to SMA/CD90-expressing myofibroblasts. We demonstrate therefore for the first time fate commitment among bone marrow-derived Schwann cells. The therapeutic potential of these cells may be tested in future transplantation studies. (206 words)., ((c) 2010 Elsevier Inc. All rights reserved.)

We examined the functional maturation of canal-related brainstem neurons in Sprague-Dawley rats at postnatal day (P)1 to adult. Conscious animals were subjected to cycles of angular acceleration and deceleration so as to selectively activate hair cells of the horizontal semicircular canals. Brainstem neurons were monitored for c-fos expression by immuno-hybridization histochemistry as an indicator of neuronal activation. Fos-immunoreactive canal-related neurons were identifiable from P4 onwards in the vestibular nucleus and downstream vestibular relay stations, prepositus hypoglossal nucleus, and inferior olive. In the vestibular nucleus and prepositus hypoglossal nucleus, the number of canal-related neurons increased progressively with age, reaching the adult level by P21. Those in the inferior olive increased in number from P4 to P14 but decreased significantly afterwards until adulthood. The topography was not clear in the vestibular nucleus and prepositus hypoglossal nucleus. Canal-related neurons in P4-7 rats were spread throughout the rostrocaudal length of each subnucleus but clusters of canal-related neurons tended to form within specific subnuclei by P21. These were concentrated in the caudal halves of medial and spinal vestibular nuclei and the rostral parts of superior vestibular nucleus and prepositus hypoglossal nucleus. In the inferior olive, the topography was evident early in the course of development. Canal-related neurons were exclusively located in four subnuclei: dorsal medial cell column, dorsal cap, subnucleus A, and subnucleus C, but not in other subnuclei. Taken together, our data revealed the developmental profile of neuronal subpopulations within the horizontal canal system, thus providing an internal neural representation for postnatal coding of horizontal head rotations in spatial perception., ((c) 2009 Wiley-Liss, Inc.)

To examine whether subgroups of vestibular nuclear neurons encode different frequency oscillation of horizontal linear motion, Fos immunohistochemistry was used to document neuronal subpopulations that were functionally activated by such otolithic stimulations. Conscious rats at P7, P14 and adult were subjected to sinusoidal linear acceleration along the transverse axis on the horizontal plane. Labyrinthectomized and/or stationary controls showed only sporadically scattered Fos-labeled neurons in the vestibular nuclei, confirming otolithic origin of c-fos expression. In each age group, Fos-labeled neurons responsive to high frequency stimulation (>1.5 Hz) were clustered in the lateral region of the medial vestibular nucleus while those to low frequency stimulation (0.5-1.0 Hz) were found in the medial portion of the medial vestibular nucleus. The number of these neurons increased with age. No apparent frequency-related distribution pattern of Fos-labeled neurons was observed in other vestibular nuclei and subgroups. Our findings therefore reveal subpopulations of central vestibular neurons responsive to different stimulus frequencies that correspond to head motions ranging from tilt to translation., (Copyright 2010 Elsevier B.V. All rights reserved.)

Persistent proteolytic imbalance in chronic inflammatory diseases has been ascribed to neutrophil elastase (NE)/antielastase imbalance in wound fluids. In sputum sols of patients with bronchiectasis, we found unopposed NE activity, despite overwhelming excess of the physiological antielastase, alpha(1)-antitrypsin (alpha(1)-AT). Western blot analysis found NE in a supramolecular complex with shed ectodomains of syndecan (Syn)-1 in sputum sol samples and, as such, inhibition of NE activity was incomplete, even with addition of exogenous alpha(1)-AT. To confirm that NE binding to heparan sulfate (HS) components of Syn-1 limits the antielastase effect, recombinant human Syn-1 was recovered from stable Syn-1 transfectants of a human B-lymphoid cell line (ARH-77). Western ligand blot confirmed that NE bound to HS moieties and alpha(1)-AT to the core protein of the recombinant product. Inhibition of NE activity by standard additions of alpha(1)-AT was incomplete unless Syn-1 had been deglycanated by heparitinase treatment. Surface plasmon resonance analysis revealed that NE binding to HS (equilibrium dissociation constant, approximately 14 nM) could be outcompeted by heparin variants. We conclude that the HS moiety of shed Syn-1 binds and restricts NE from inhibition by alpha(1)-AT.

Expression of c-fos in the medial geniculate body (MGB) and the inferior colliculus (IC) in response to bicuculline-induced corticofugal activation was examined in rats at different time points after bilateral cochlear ablation (4 h-30 days). Corticofugal activation was crucial in eliciting Fos expression in the MGB after cochlear ablation. The pars ovoidea (OV) of the medial geniculate body ventral division (MGv) showed dense Fos expression 4 h after cochlear ablation; the expression declined to very low levels at 24 h and thereafter. In turn, strong Fos expression was found in the pars lateralis (LV) of the MGv 24 h after cochlear ablation and dropped dramatically at 14 days. The dorsal division of the MGB (MGd) showed high Fos expression 7 days after cochlear ablation, which persisted for a period of time. Using multi-electrode recordings, neuronal activity of different MGB subnuclei was found to correlate well with Fos expressions. The temporal changes in cortically activated Fos expression in different MGB subnuclei after bilateral cochlear ablation indicate differential denervation hypersensitivities of these MGB neurons and likely point to differential dependence of these nuclei on both auditory ascending and corticofugal descending inputs. After bilateral cochlear ablation, significant increases in Fos-positive neurons were detected unilaterally in all IC subnuclei, ipsilateral to the bicuculline injection.

This review focuses on our effort in addressing the development and lesion-induced plasticity of the gravity sensing system. After severance of sensory input from one inner ear, there is a bilateral imbalance in response dynamics and spatial coding behavior between neuronal subpopulations on the two sides. These data provide the basis for deranged spatial coding and motor deficits accompanying unilateral labyrinthectomy. Recent studies have also confirmed that both glutamate receptors and neurotrophin receptors within the bilateral vestibular nuclei are implicated in the plasticity during vestibular compensation and development. Changes in plasticity not only provide insight into the formation of a spatial map and recovery of vestibular function but also on the design of drugs for therapeutic strategies applicable to infants or vestibular disorders such as vertigo and dizziness.

Growing evidence has shown that the p75 neurotrophin receptor (p75NTR) may play important roles in controlling neuronal survival or cell apoptosis within the central nervous system in development, and in pathological or neural injury. Recent studies have further revealed that p75NTR acts as a "molecular signal switch" that determines cell death or survival by three processes. First, pro-nerve growth factor (proNGF) triggers cell apoptosis by its high affinity binding to p75NTR, while NGF induces neuronal survival with low-affinity binding. Second, p75NTR mediates cell death by combining with co-receptor sortilin, whereas it promotes neuronal survival through combination with proNGF. Third, release of the intracellular domain chopper or cleavaged "short p75NTR" can independently initiate neuronal apoptosis. We have identified the cell self-destructive proNGF-p75NTR-sortilin signalling apparatus assembled in ventral tier dopamine neurons of the substantia nigra pars compacta, suggesting that p75NTR signalling might be involved in selective cell death mechanisms of substantia nigra neurons or disease progression of Parkinson's disease (PD). In addition, experimental manipulation of p75NTR benefited cell survival of cholinergic or motor neurons and improved disease progression of the neurodegenerative diseases Alzheimer's disease and Amyotrophic lateral sclerosis. The proNGF-p75NTR-sortilin signalling complex may thus provide new target for neuroprotection of substantia nigra neurons and the therapeutic treatment of PD.

We investigated the maturation profile of subunits of ionotropic glutamate receptors in vestibular nuclear neurons that were activated by sinusoidal linear acceleration along the vertical plane. The otolithic origin of Fos expression in these neurons was confirmed as a marker of functional activation when labyrinthectomized and/or stationary control rats contrasted by showing sporadically scattered Fos-labeled neurons in the vestibular nuclei. By double immunohistochemistry for Fos and one of the receptor subunits, otolith-related neurons that expressed either alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate or N-methyl-d-aspartate subunits were first identified in the medial vestibular nucleus, spinal vestibular nucleus and Group x by postnatal day (P)7, and in the lateral vestibular nucleus and Group y by P9. No double-labeled neurons were found in the superior vestibular nucleus. Within each vestibular subnucleus, these double-labeled neurons constituted approximately 90% of the total Fos-labeled neurons. The percentage of Fos-labeled neurons expressing the GluR1 or NR2A subunit showed developmental invariance in all subnuclei. For Fos-labeled neurons expressing the NR1 subunit, similar invariance was observed except that, in Group y, these neurons decreased from P14 onwards. For Fos-labeled neurons expressing the GluR2, GluR2/3, GluR4 or NR2B subunit, a significant decrease was found by the adult stage. In particular, those expressing the GluR4 subunit showed a two- to threefold decrease in the medial vestibular nucleus, spinal vestibular nucleus and Group y. Also, those expressing the NR2B subunit showed a twofold decrease in Group y. Taken together, the postsynaptic expression of ionotropic glutamate receptor subunits in different vestibular subnuclei suggests that glutamatergic transmission within subregions plays differential developmental roles in the coding of gravity-related vertical spatial information.

We examined the expression profile of subunits of ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off-vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith-related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos-immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double-immunohistochemical data were corroborated by combined immuno-/hybridization histochemical data obtained from Fos-immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos-immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos-immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos-immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity-related horizontal head movements., ((c) 2008 Wiley-Liss, Inc.)

This immunolocalization study was performed to investigate the temporal and spatial expression of tissue inhibitors of metalloproteinase (TIMP) 1 within endochondral and intramembranous bone grafts during the early stages of healing, in the hope of gaining a better understanding of the mechanisms of bone graft healing, which could influence the choice of bone graft used. Twenty-seven adult New Zealand White rabbits were used as the experimental model. Autogenous bone grafts taken from the cranial bone (intramembranous in origin) and the femur (endochondral in origin) were grafted into skull defects created on either side of the parietal suture. Rabbits were killed on days 1 to 9 postgrafting, and the bone graft alone was harvested for immunolocalization of TIMP-1. In endochondral bone grafts, TIMP-1 was expressed on days 1 to 3, followed by a period of absence until days 8 and 9. Intramembranous bone grafts did not express TIMP-1 until days 6 to 9. The timing and location of TIMP-1 expression coincided with osteogenesis, which indicates a role for TIMP-1 in preserving newly formed bone during the initial stages of graft healing. The differential temporal expression of TIMP-1 in endochondral and intramembranous bone grafts suggests that bone graft type plays an important role in influencing the healing process mediated by the host tissues. The earlier expression of TIMP-1 in endochondral bone grafts could be the reason for delayed vascularization of defects containing these grafts, whereas the delayed expression of TIMP-1 in intramembranous bone grafts could allow earlier vascularization of the intramembranous bone grafts.

The airway epithelium participates in chronic airway inflammation by expressing adhesion molecules that mediate the transmigration of neutrophils into the inflamed airways. We hypothesize that, in patients with bronchiectasis, cytokines in their bronchial secretions enhance the expression of intercellular cell adhesion molecule (ICAM-1) in the bronchial epithelium and thus contribute to sustained recruitment of neutrophils into the inflamed airways. In the present study, we investigated the effect of bronchial secretions on the regulation of ICAM-1 in bronchial epithelial cells, and its modulation by pharmacologic agents. The expression of ICAM-1 mRNA and protein in human bronchial epithelial cells upon exposure to sputum sol from subjects with bronchiectasis were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, respectively. Modulating effects of dexamethasone, ibuprofen, MK-663 or triptolide on ICAM-1 regulation were investigated in vitro. We demonstrated that changes in ICAM-1 expression correlated with levels of TNF-alpha in the sputum sol, and treatment of sol samples with TNF-alpha antibodies attenuated both the increase in ICAM-1 mRNA and protein. The role of TNF-alpha was further demonstrated when TNF-alpha elicited dose dependent increase in ICAM-1 expression. The sputum effect could also be suppressed dose-dependently by pre-incubation of bronchial epithelial cells with dexamethasone, ibuprofen, MK-663 or triptolide. Evidence is thus provided for the upregulation of bronchial epithelial ICAM-1 expression by airway secretions in bronchiectasis and a specific role for TNF-alpha in the secretions. The success of drug attenuation of this upregulation provides insight into possible therapeutic paradigms in the management of the disease.

To investigate the corticofugal modulation of acoustic information ascending through the auditory pathway of the rat, immunohistochemical techniques were used to study the functional expression of Fos protein in neurons. With auditory stimulation at different frequencies, Fos expression in the medial geniculate body (MGB), inferior colliculus (IC), superior olivary complex, and cochlear nucleus was examined, and the extent of Fos expression on the two sides was compared. Strikingly, we found densely Fos-labeled neurons in all divisions of the MGB after both presentation of an auditory stimulus and administration of a gamma-aminobutyric acid type A (GABA(A)) antagonist (bicuculline methobromide; BIM) to the auditory cortex. The location of Fos-labeled neurons in the ventral division (MGv) after acoustic stimulation at different frequencies was in agreement with the known tonotopic organization. That no Fos-labeled neurons were found in the MGv with acoustic stimuli alone suggests that the transmission of ascending thalamocortical information is critically governed by corticofugal modulation. The dorsal (DCIC) and external cortices (ECIC) of the IC ipsilateral to the BIM-injected cortex showed a significantly higher number of Fos-labeled neurons than the contralateral IC. However, no difference in the number of Fos-labeled neurons was found between the central nucleus of the IC on either side, indicating that direct corticofugal modulation occurs only in the ECIC and DCIC. Further investigations are needed to assess the functional implications of the morphological differences observed between the descending corticofugal projections to the thalamus and the IC., ((c) 2007 Wiley-Liss, Inc.)

Urinary polyanions recovered from the urine samples of kidney stone-formers and normal controls were subjected to preparative agarose gel electrophoresis, which yielded fractions 1-5 in a decreasing order of mobility. In both groups, chondroitin sulfates were identified in the fast-moving fractions and heparan sulfates in the slow-moving fractions. Furthermore, two types of heparan sulfates were identified based on their electrophoretic mobility: slow-moving and fast-moving. The fractionated urinary polyanions were then tested in an in vitro calcium oxalate crystallization assay and compared at the same uronic acid concentration, whereby, the chondroitin sulfates of stone-formers and heparan sulfates of normals enhanced crystal nucleation. Fraction 5 of the normals, containing glycoproteins (14-97 kDa) and associated glycosaminoglycans, were found to effectively inhibit crystallization. Papainization of this fraction in stone-formers revealed crystal-suppressive effects of glycoproteins, which was not seen in similar fractions of normals. It was concluded that glycoproteins could modulate the crystal-enhancing glycosaminoglycans such as chondroitin sulfates of stone-formers but not in normals. The differing crystallization activities of electrophoretic fraction 1 of normals and stone-formers revealed the presence of another class of glycosaminoglycan-hyaluronan. Hence, in the natural milieu, different macromolecules combine to have an overall outcome in the crystallization of calcium oxalate.

This review focuses on recent approaches to unravel the capacity of otolith-related brainstem neurons for coding head orientations. In the first section, the spatiotemporal features of central vestibular neurons in response to natural otolithic stimulation are reviewed. Experiments that reveal convergent inputs from bilateral vestibular end organs bear important implications on the processing of spatiotemporal signals and integration of head orientational signals within central otolith neurons. Another section covers the maturation profile of central otolith neurons in the recognition of spatial information. Postnatal changes in the distribution pattern of neuronal subpopulations that subserve the horizontal and vertical otolith systems are highlighted. Lastly, the expression pattern of glutamate receptor subunits and neurotrophin receptors in otolith-related neurons within the vestibular nuclear complex are reviewed in relation to the potential roles of these receptors in the development of vestibular function.

To investigate the critical maturation time of otolith-related neurons in processing vertical orientations, rats (postnatal day 4 to adults) were studied for functional activation of c-fos expression in brainstem neurons by immuno-/hybridization histochemistry. Conscious rats were subjected to sinusoidal linear acceleration along the vertical plane. Labyrinthectomized and/or stationary controls showed only sporadically scattered Fos-labeled neurons in the vestibular nuclei, confirming an otolithic origin of c-fos expression. Functionally activated Fos expression in neurons of the medial and spinal vestibular nuclei and group x were identifiable by P7 and those in group y by P9. A small number of Fos-labeled neurons characterized by small soma size were found in the ventral part of lateral vestibular nucleus by P9. Other vestibular-related areas such as prepostitus hypoglossal nucleus, gigantocellular reticular nucleus and locus coeruleus of normal experimental rats showed functionally activated c-fos expression at P7. Neurons in dorsal medial cell column and beta subnucleus of the inferior olive only showed functionally activated c-fos expression by the second postnatal week. These findings revealed a unique critical maturation time for each of the vestibular-related brainstem areas in the recognition of gravity-related vertical head orientations. By mapping the three-dimensional distribution of Fos-immunoreactive neurons, we found an even distribution of otolith-related neurons within the spinal vestibular nucleus in groups x and y but a clustered distribution in the middle-lateral-ventral part of the medial vestibular nucleus. Taken together, our findings reveal the developmental profile of neuronal subpopulations within the vertical otolith system, thereby providing an anatomical basis for postnatal coding of gravity-related vertical head movements.

The aim of this study was to investigate the temporal expression of TIMP-1 within endochondral and intramembranous bone grafts during the early stages of healing in thirty six adult New Zealand white rabbits. Total RNA was isolated from bone grafts extracted on days 0-11 and day 14 post-grafting, for RT-PCR analysis. In situ hybridization was carried out on days 1-9 and day 14. Results showed TIMP-1 expression coincides with osteogenesis, which indicates a role for TIMP in preserving the newly formed bone during the initial stages of graft healing. Bone grafts play an important role in influencing the healing process mediated by the host tissues. The temporal expression of TIMP-1 differs in endochondral and intramembranous bone grafts. The earlier expression of TIMP-1 by endochondral bone grafts, could be the reason for the delayed vascularization while the expression of TIMP-1 by the intramembranous bone grafts, at a later stage could allow for earlier vascularization.

Cell migration is central to development and post-traumatic regeneration. The differential increase in 6-sulphated chondroitins during axonal growth in both crushed sciatic nerves and brain development suggests that chondroitin 6-sulphotransferase-1 (C6ST-1) is a key enzyme that mediates cell migration in the process. We have cloned the cDNA of the C6ST-1 gene (C6st1) (GenBank accession number AF178689) from crushed sciatic nerves of adult rats and produced ribonucleotide probes accordingly to track signs of 6-sulphated chondroitins at the site of injury. We found C6st1 mRNA expression in Schwann cells emigrating from explants of both sciatic nerve segments and embryonic dorsal root ganglia. Immunocytochemistry indicated pericellular 6-sulphated chondroitin products around C6ST-1-expressing frontier cells. Motility analysis of frontier cells in cultures subjected to staged treatment with chondroitinase ABC indicated that freshly produced 6-sulphated chondroitin moieties facilitated Schwann cell motility, unlike restrictions resulting from proteoglycan interaction with matrix components. Sciatic nerve crush provided further evidence of in vivo upregulation of the C6ST-1 gene in mobile Schwann cells that guided axonal regrowth 1-14 days post crush; downregulation then accompanied declining mobility of Schwann cells as they engaged in the myelination of re-growing axons. These findings are the first to identify upregulated C6st1 gene expression correlating with the motility of Schwann cells that guide growing axons through both developmental and injured environments.