Quantitative analysis of electrophysiologic data during recovery.

<p><i>Top row</i>, Amplitude (in µV) of the somatosensory evoked potentials detected over the S1_FL cortical area of the brain, acquired during the fMRI sessions. <i>Bottom row</i>, result of the sticky tape behavioral test. Time to contact the tape stuck to the forepaws of the animals is presented (mean ± standard deviation, in seconds). As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012779#pone-0012779-g004" target="_blank">Fig. 4</a>, the plots present the data for the control group (white bars), and for the group of animals treated with stem cells that recovered BOLD signal (dark gray bars).</p

Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies

<div><p>Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (iv, ip, sc), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000–300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.</p> </div

The photon flux maximum and timing are dependent on the route of substrate administration.

<p>a, b) The PE_max and AUC increase corresponding to the physiologically expected biodistribution for sc, ip and iv substrate administration, reflected by the characteristic time activity curves. c) Maximal photon flux is reached at minimal time for iv injections followed by ip and sc (* statistically significant difference with p≤0.05 to standard protocol 150 mg/kg post-Iso in post hoc comparison with Sidak correction).</p

Functional brain recovery during six months observation.

<p>BOLD images obtained during the 24 weeks exploration period for two control animals (top rows), presenting a permanent loss of BOLD signal on the affected right hemisphere, and the two stem cell implanted animals (bottom rows) that regained BOLD activity at the affected hemisphere starting from week 10.</p

Photon flux is modulated by the type of anesthesia.

<p>a) PE_max and AUC were decreased under Ketamine/Xylazine conditions but not different for Pentobarbital compared to Isoflurane. b, c) Representative time activity curves showing the anesthesia dependent signal behavior leading to delayed time-to-peak for Ketamine/Xylazine, but no clear difference between Isoflurane and Pentobarbital anesthesia. (* statistically significant difference with p≤0.05 to standard protocol 150 mg/kg post-Iso in post hoc comparison with Sidak correction).</p

Luciferase inhibition by Isoflurane is avoided by substrate administration before anesthesia onset.

<p>a, b) Difference in PE_max and AUC under pre/post Isoflurane conditions becomes more pronounced with increasing substrate concentration. c) The order of application between Isoflurane anesthesia and substrate had no impact on the time-to-peak for 15, 150 and 300 mg/kg D-Luciferin. (* statistically significant difference with p≤0.05 to standard protocol 150 mg/kg post-Iso in post hoc comparison with Sidak correction;+statistically significant difference with p≤0.05 between pre and post condition in post hoc comparison with Sidak correction).</p

Selection process of the animals for the treatment inclusion.

<p>Animals were selected according to the outcome from the MCAO surgery (only subcortical vs. cortico-subcortical lesions) and the fMRI study performed at week 3 (regained vs. non-regained BOLD activation). Finally the remaining animals were randomly asigned to a control and a stem cell-treated group. Gray boxes indicate animals excluded from further procedures at each time point, while white boxes indicate the animals selected for successive stages of the study.</p

Photon emission is substantially increased by the modified BLI protocol.

<p>a) Representative images (equally scaled) for each cell number grafted into nude mice acquired with the standard protocol (upper row) and with the advanced protocol (lower row) reveal the objective sensitivity benefit, which is also represented in the quantitative SNR values. b) Correlation between photon emission and cell number revealed a linear relationship under in vivo conditions, with a steeper slope for the novel protocol, indicating the increased sensitivity.</p

Localization of labeled cells by T2*-weighted MRI.

<p>3D high resolution T2*-weighted MR images, pre- (1st column) and post- (2nd column) implantation of USPIO-labeled stem cells, showing the successful implantation of the cells into the brain parenchyma. Corresponding T2-weighted images (3^rd column) confirm that cells were implanted in healthy tissue.</p

Characterization of Luc2-expressing NSCs.

<p>a) Efficient NSC transduction and selection process (by FACS and antibiotics) was confirmed by the homogeneous expression of the fluorescent reporter copGFP, which directly reflects Luc2 expression because of the T2A linker element (microscopic images 20× magnification, 50 µm scale bar). b) Reporter gene expression had no impact on cell viability, as confirmed with the PrestoBlue assay (data of 5 independent measurements presented as relative fluorescent units, RFU). c, d) Quantitative analysis of NSC<i>^Luc2+</i> dilution series (1 min acquisition at 37°C with 30 µg/ml D-Luciferin) revealed a linear correlation between photon emission and cell number, as well as SNR in vitro.</p