Grégory Hans

Plasticity of Cultured Mesenchymal Stem Cells: Switch from Nestin‐Positive to Excitable Neuron‐Like Phenotype

Bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but, under appropriate experimental conditions, can also differentiate into nonmesenchymal cells—for instance, neural cells. These observations have raised interest in the possible use of MSCs in cell therapy strategies for various neurological disorders. In the study reported here, we addressed the question of in vitro differentiation of MSCs into functional neurons. First, we demonstrate that when they are co‐cultured with cerebellar granule neurons, adult MSCs can express neuronal markers. Two factors are needed for the emergence of neuronal differentiation of the MSCs: the first one is nestin expression by MSCs (nestin is a marker for the responsive character of MSCs to extrinsic signals), and the second one is a direct cell–cell interaction between neural cells and MSCs that allows the integration of these extrinsic signals. Three different approaches suggest that neural phenotypes arise from MSCs by a differentiation rather than a cell fusion process, although this last phenomenon can also coexist. The expression of several genes—including sox, pax, notch, delta, frizzled, and erbB—was analyzed by quantitative reverse transcription polymerase chain reaction (RT‐PCR) in order to further characterize the nestin‐positive phenotype compared to the nestin‐negative one. An overexpression of sox2, sox10, pax6, fzd, erbB2, and erbB4 is found in nestin‐positive MSCs. Finally, electrophysiological analyses demonstrate that MSC‐derived neuron‐like cells can fire single‐action potentials and respond to several neurotransmitters such as GABA, glycine, and glutamate. We conclude that nestin‐positive MSCs can differentiate in vitro into excitable neuron‐like cells.

The anti-epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA- and glycine-gated currents

In this study in vitro and in vivo approaches were combined in order to investigate if the anti‐epileptic mechanism(s) of action of levetiracetam (LEV; Keppra®) may involve modulation of inhibitory neurotransmission. GABA‐ and glycine‐gated currents were studied in vitro using whole‐cell patch‐clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound‐susceptible mice. The effect of LEV was compared with reference anti‐epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide. LEV contrasted the reference AEDs by an absence of any direct effect on glycine‐gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA‐gated currents. A similar action on GABA‐elicited currents was observed with the reference AEDs, except ethosuximide. These minor direct effects contrasted with a potent ability of LEV (EC50=1 – 10 μM) to reverse the inhibitory effects of the negative allosteric modulators zinc and β‐carbolines on both GABAA and glycine receptor‐mediated responses. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of β‐carbolines on GABA‐gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine‐gated currents and only clonazepam and phenobarbital inhibited the action of DMCM. LEV (17 mg kg−1) produced a potent suppression of sound‐induced clonic convulsions in mice. This protective effect was significantly abolished by co‐administration of the β‐carboline FG 7142, from a dose of 5 mg kg−1. In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg−1) was without any effect on the protection afforded by LEV. The results of the present study suggest that a novel ability to oppose the action of negative modulators on the two main inhibitory ionotropic receptors may be of relevance for the anti‐epileptic mechanism(s) of action of LEV.

Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4.

BackgroundSpontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinsons disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies.ResultsIn this study, we addressed the question of the possible influence of mesenchymal stem cells on neural stem cell fate. We have previously reported that adult rat mesenchymal stem cells are able to express nestin in defined culture conditions (in the absence of serum and after 25 cell population doublings) and we report here that nestin-positive (but not nestin-negative) mesenchymal stem cells are able to favour the astroglial lineage in neural progenitors and stem cells cultivated from embryonic striatum. The increase of the number of GFAP-positive cells is associated with a significant decrease of the number of Tuj1- and O4-positive cells. Using quantitative RT-PCR, we demonstrate that mesenchymal stem cells express LIF, CNTF, BMP2 and BMP4 mRNAs, four cytokines known to play a role in astroglial fate decision. In this model, BMP4 is responsible for the astroglial stimulation and oligodendroglial inhibition, as 1) this cytokine is present in a biologically-active form only in nestin-positive mesenchymal stem cells conditioned medium and 2) anti-BMP4 antibodies inhibit the nestin-positive mesenchymal stem cells conditioned medium inducing effect on astrogliogenesis.ConclusionsWhen thinking carefully about mesenchymal stem cells as candidates for cellular therapy in neurological diseases, their effects on resident neural cell fate have to be considered.

Substance P protects spiral ganglion neurons from apoptosis via PKC-Ca2+-MAPK/ERK pathways

In the current study, we have investigated the ability of substance P (SP) to protect 3‐day‐old (P3) rat spiral ganglion neurons (SGNs) from trophic factor deprivation (TFD)‐induced cell death. The presence of SP high affinity neurokinin‐1 receptor (NK1) transcripts was detected in the spiral ganglion and the NK1 protein localized to SGNs both ex vivo and in vitro. Treatment with SP increased cytoplasmic Ca2+ in SGNs, further arguing for the presence of functional NK1 on these neurons. Both SP and the agonist [Sar9,Met(O2)11]‐SP significantly decreased SGN cell death induced by TFD, with no effect on neurite outgrowth. The survival promoting effect of SP was blocked by the NK1 antagonist, WIN51708. Both pan‐caspase inhibitor BOC‐D‐FMK and SP treatments markedly reduced activation of caspases and DNA fragmentation in trophic factor deprived‐neurons. The neuroprotective action of SP was antagonised by specific inhibitors of second messengers, including 1.2‐bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM) to chelate cytosolic Ca2+, the protein kinase C (PKC) inhibitors bisindolylmaleimide I, Gö6976 and LY333531 and the MAPK/ERK inhibitor U0126. In contrast, nifedipine, a specific inhibitor of l‐type Ca2+ channel, and LY294002, a phosphatidylinositol‐3‐OH kinase (PI3K) inhibitor, had no effect on SP trophic support of SGNs. Moreover, activation of endogenous PKC by 4β‐phorbol 12‐myristate 13‐acetate (PMA) also reduced the loss of trophic factor‐deprived SGNs. Thus, NK1 expressed by SGNs transmit a survival‐promoting regulatory signal during TFD‐induced SGN cell death via pathways involving PKC activation, Ca2+ signalling and MAPK/ERK activation, which can be accounted for by an inhibition of caspase activation.

Comparison of the Surgical Pleth Index™ with haemodynamic variables to assess nociception–anti-nociception balance during general anaesthesia

BACKGROUND The Surgical Pleth Index (SPI) is proposed as a means to assess the balance between noxious stimulation and the anti-nociceptive effects of anaesthesia. In this study, we compared SPI, mean arterial pressure (MAP), and heart rate (HR) as a means of assessing this balance. METHODS We studied a standard stimulus [head-holder insertion (HHI)] and varying remifentanil concentrations (CeREMI) in a group of patients undergoing neurosurgery. Patients receiving target-controlled infusions were randomly assigned to one of the three CeREMI (2, 4, or 6 ng m⁻¹), whereas propofol target was fixed at 3 µg ml⁻¹. Steady state for both targets was achieved before HHI. Intravascular volume status (IVS) was evaluated using respiratory variations in arterial pressure. Prediction probability (Pk) and ordinal regression were used to assess SPI, MAP, and HR performance at indicating CeREMI, and the influence of IVS and chronic treatment for high arterial pressure, as possible confounding factors. RESULTS The maximum SPI, MAP, or HR observed after HHI correctly indicated CeREMI in one of the two patients [accurate prediction rate (APR)=0.5]. When IVS and chronic treatment for high arterial pressure were taken into account, the APR was 0.6 for each individual variable and 0.8 when all of them predicted the same CeREMI. That increase in APR paralleled an increase in Pk from 0.63 to 0.89. CONCLUSIONS SPI, HR, and MAP are of comparable value at gauging noxious stimulation-CeREMI balance. Their interpretation is improved by taking account of IVS, treatment for chronic high arterial pressure, and concordance between their predictions.

Glycine triggers an intracellular calcium influx in oligodendrocyte progenitor cells which is mediated by the activation of both the ionotropic glycine receptor and Na+‐dependent transporters

Using fluo‐3 calcium imaging, we demonstrate that glycine induces an increase in intracellular calcium concentration ([Ca2+]i) in cortical oligodendrocyte progenitor (OP) cells. This effect results from a calcium entry through voltage‐gated calcium channels (VGCC), as it is observed only in OP cells expressing such channels, and it is abolished either by removal of calcium from the extracellular medium or by application of an l‐type VGCC blocker. Glycine‐triggered Ca2+ influx in OP cells actually results from an initial depolarization that is the consequence of the activation of both the ionotropic glycine receptor (GlyR) and Na+‐dependent transporters, most probably the glycine transporters 1 (GLYT1) and/or 2 (GLYT2) which are colocalized in these cells. Through this GlyR‐ and transporter‐mediated effect on OP intrcellular calcium concentration [Ca2+]i, glycine released by neurons may, as well as other neurotransmitters, serve as a signal between neurons and OP during development.

Activation of protein kinase CbetaI constitutes a new neurotrophic pathway for deafferented spiral ganglion neurons.

In mammals, degeneration of peripheral auditory neurons constitutes one of the main causes of sensorineural hearing loss. Unfortunately, to date, pharmacological interventions aimed at counteracting this condition have not presented complete effectiveness in protecting the integrity of cochlear neural elements. In this context, the protein kinase C (PKC) family of enzymes are important signalling molecules that play a role in preventing neurodegeneration after nervous system injury. The present study demonstrates, for the first time, that the PKC signalling pathway is directly neurotrophic to axotomised spiral ganglion neurons (SGNs). We found that PKCβI was strictly expressed by postnatal and adult SGNs both in situ and in vitro. In cultures of SGNs, we observed that activators of PKC, such as phorbol esters and bryostatin 1, induced neuronal survival and neurite regrowth in a manner dependent on the activation of PKCβI. The neuroprotective effects of PKC activators were suppressed by pre-treatment with LY294002 (a PI3K inhibitor) and with U0126 (a MEK inhibitor), indicating that PKC activators promote the survival and neurite outgrowth of SGNs by both PI3K/Akt and MEK/ERK-dependent mechanisms. In addition, whereas combining the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) was shown to provide only an additive effect on SGN survival, the interaction between PKC and neurotrophin signalling gave rise to a synergistic increase in SGN survival. Taken together, the data indicate that PKCβI activation represents a key factor for the protection of the integrity of neural elements in the cochlea.

Identification of factors that maintain mammalian outer hair cells in adult organ of Corti explants

Both outer hair cells (OHCs) and inner hair cells (IHCs) survive and mature in 3 days old rat organ of Corti explants cultured for 1 month in a minimal essential medium. In contrast, under the same culture conditions, only IHCs survive in explants from adult guinea pig organ of Corti while many of the OHCs are lost within the first 48 h. Hair cell counts show OHCs loss to be greater in the lower portion (i.e. middle turn) of the cochlea than at the apex. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) indicates that there is DNA damage in adult OHCs within 8 h of explantation. Treatment of the adult organ of Corti explants with either actinomycin D (10(-7) M) or cycloheximide (10(-6) M) prevents most OHC losses. According to these results apoptosis may be the mechanism of OHC loss in adult organ of Corti explants. Stable membrane potentials recorded from the OHCs in both uncultured and actinomycin D-treated organ of Corti explants cultured for 72 h demonstrate the functional integrity of these hair cells. OHC losses in the adult guinea pig organ of Corti cultures can also be prevented by treatment with several of the growth factors tested, i.e. acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), transforming growth factor-beta1 (TGF-beta1), and glial cell-derived neurotrophic factor (GDNF). The results of this study suggest that growth factor therapy may be applicable to the treatment of some hearing disorders.

The place of viscoelastic testing in clinical practice

Hellmut Hartert was the first person to exploit the viscoelastic properties of clotting blood to measure blood coagulation in 1948. Since then, the technology has improved, allowing these analyses to be performed as point‐of‐care tests with immediately‐available results. The addition of several activators and inhibitors to the original assay creates a panel of tests able to quantify the different aspects of blood clotting that can rival conventional laboratory assays. However, although much progress has been made, the standardization and validation of these tests still need improvement. Viscoelastic analyses of blood coagulation are mainly used to guide haemostatic therapy in bleeding patients and have proven superior to standard clotting tests in some circumstances. There is potential to extend their use to other areas, such as drug monitoring, and diagnosis and management of congenital bleeding disorders. The forthcoming cartridge‐based assays are expected to improve the reliability and usability of viscoelastic assays of blood coagulation but high quality clinical trials remain urgently needed to determine their exact place, benefit and cost effectiveness.

Intravenous lidocaine infusion reduces bispectral index-guided requirements of propofol only during surgical stimulation

BACKGROUND I.V. lidocaine reduces volatile anaesthetics requirements during surgery. We hypothesized that lidocaine would also reduce propofol requirements during i.v. anaesthesia. METHODS A randomized controlled study of 40 patients tested the effect of i.v. lidocaine (1.5 mg kg(-1) then 2 mg kg(-1) h(-1)) on propofol requirements. Anaesthesia was maintained with remifentanil and propofol target-controlled infusions (TCI) to keep the bispectral index (BIS) around 50. Effect-site concentrations of propofol and remifentanil and BIS values were recorded before and after skin incision. Data were analysed using anova and mixed effects analysis with NONMEM. Two dose-response studies were then performed with and without surgical stimulation. Propofol TCI titrated to obtain a BIS around 50 was kept constant. Then patients were randomized into four groups: A, saline; B, 0.75 mg kg(-1) bolus then infusion 1 mg kg(-1) h(-1); C, 1.5 mg kg(-1) bolus and infusion 2 mg kg(-1) h(-1); and D, 3 mg kg(-1) bolus and infusion 4 mg kg(-1) h(-1). Lidocaine administration coincided with skin incision. BIS values and haemodynamic variables were recorded. Data were analysed using linear regression and two-way anova. RESULTS Lidocaine decreased propofol requirements (P<0.05) only during surgery. In the absence of surgical stimulation, lidocaine did not affect BIS nor haemodynamic variables, whereas it reduced BIS increase (P=0.036) and haemodynamic response (P=0.006) secondary to surgery. CONCLUSIONS The sparing effect of lidocaine on anaesthetic requirements seems to be mediated by an anti-nociceptive action.