Automated aspiration of stem cell colony fractions from feeder cell monolayers.

<p>Panel i.–v.: Automated documentation of a representative aspiration procedure using the CellCelector™/Soft Imaging System Cell^D. lane i.–v.: Determination of feeder cell contamination by RT-PCR expression analysis of nr mRNA.</p

Analysis of feeder cell contamination during neuronal differentiation of murine embryonic stem cells.

<p>Feeder cells were transfected with the neomycine resistance (<i>nr</i>) gene. A. RT-CR and nested RT-PCR analysis of <i>nr</i> gene expression (NR) during neuronal differentiation of murine ESCs. Displayed are images of representative results following RT-PCR analysis. GAPDH was used as a control. B. Percentage of feeder cell contamination was evaluated by <i>nr</i> gene expression analysis of predefined ratios of stem and feeder cells. Displayed are representative results of PCR products. C. Following logarithmic transformation signal density is plotted as a linear function against feeder cell number (n = 4).</p

Protein expression analysis of neuronal differentiation markers.

<p>Immunocytochemical analysis of microtubulin associated protein 2 (<i>MAP2;</i> cyctoplasma, green), neuron-specific protein (<i>NeuN;</i> nucleus, blue), and neuron-specific enolase (<i>NSE</i>; cytoplasam, red) at phase V in <i>f-f</i>SCs and <i>st</i>SCs.</p

Gene expression analysis of neuronal differentiation markers by semi-quantitative RT-PCR.

<p>A. Displayed are images of representative results following RT-PCR analysis. B. Quantitative analysis of neuronal and cell lineage marker expression in feeder-freed stem cells (<i>f-f</i>) and stem cells grown under standard conditions (<i>st</i>) (±SEM, n = 6). <i>grey: st</i>SCs; black: <i>f-f</i>SCs.</p

Quantitative analysis of time dependent increase in cross sectional dimensions of <i>f-f</i>EBs and <i>st</i>EBs (±SEM; n = 20).

<p><i>diamant</i>: <i>f-f</i>EBs; <i>triangle</i>: <i>st</i>EBs in hanging drops; <i>square</i>: <i>st</i>EBs in V-96 wells.</p

Protein expression analysis of neuronal or cardiomyocyte differentiation markers.

<p>A. GFP expression in <i>f-f</i>EBs and <i>st</i>EBS at day 14 (GFP under the control of αMHC-promoter). B. Immunocytochemical analysis of nestin expression at phase V in <i>f-f</i>SCs and <i>st</i>SCs.</p

Repetitive Long-Term Hyperbaric Oxygen Treatment (HBOT) Administered after Experimental Traumatic Brain Injury in Rats Induces Significant Remyelination and a Recovery of Sensorimotor Function

<div><p>Cells in the central nervous system rely almost exclusively on aerobic metabolism. Oxygen deprivation, such as injury-associated ischemia, results in detrimental apoptotic and necrotic cell loss. There is evidence that repetitive hyperbaric oxygen therapy (HBOT) improves outcomes in traumatic brain-injured patients. However, there are no experimental studies investigating the mechanism of repetitive long-term HBOT treatment-associated protective effects. We have therefore analysed the effect of long-term repetitive HBOT treatment on brain trauma-associated cerebral modulations using the lateral fluid percussion model for rats. Trauma-associated neurological impairment regressed significantly in the group of HBO-treated animals within three weeks post trauma. Evaluation of somatosensory-evoked potentials indicated a possible remyelination of neurons in the injured hemisphere following HBOT. This presumption was confirmed by a pronounced increase in myelin basic protein isoforms, PLP expression as well as an increase in myelin following three weeks of repetitive HBO treatment. Our results indicate that protective long-term HBOT effects following brain injury is mediated by a pronounced remyelination in the ipsilateral injured cortex as substantiated by the associated recovery of sensorimotor function.</p></div

Grouping of animals and number of animals per group and testing parameter.

<p>*Uneven number of animals is due to trauma- or anaesthesia-related loss of rats. ^#Different groups were implemented since this allowed for simultaneous analysis of these parameters. Biases in the results of the distinct testing parameters due to previous handling of the animals were thereby avoided, i.e. anaesthetics and interventions during MRI or SSEP did not affect behavioural potentials of the rats and vice versa. mTBI: moderate traumatic brain injury; sTBI: severe traumatic brain injury.</p

Modulation of myelin in the ipsilateral cortex at day 22 following induction of traumatic brain injury and HBO treatment.

<p>A. Quantitative analysis of myelin stained by Luxol Fast Blue. The ipsilateral hemisphere was analysed as a whole in order to avoid bias. Representation of the % tissue translucence of the ipsilateral hemisphere as compared to sham controls; a minimum of 4 successive brain slices per animal were analysed, *<i>p<0.05</i>, HBO-treated versus untreated animals; B. Exemplification of proteolipid protein (PLP) staining at day 22 following induction of traumatic brain injury and HBO treatment; number of animals see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097750#pone-0097750-t001" target="_blank">Table 1</a>. Error bars represent ± standard error means.</p

Schematic representation of the schedule of HBO treatments and associated analytical assays following induction of traumatic brain injury by fluid percussion.

<p>Schematic representation of the schedule of HBO treatments and associated analytical assays following induction of traumatic brain injury by fluid percussion.</p