Optogenetic Activation of Peripheral Somatosensory Neurons in Transgenic Mice as a Neuropathic Pain Model for Assessing the Therapeutic Efficacy of Analgesics.

Optogenetics is a novel biotechnology widely used to precisely manipulate a specific peripheral sensory neuron or neural circuit. However, the use of optogenetics to assess the therapeutic efficacy of analgesics is elusive. In this study, we generated a transgenic mouse stain in which all primary somatosensory neurons can be optogenetically activated to mimic neuronal hyperactivation in the neuropathic pain state for the assessment of analgesic effects of drugs. A transgenic mouse was generated using the advillin-Cre line mated with the Ai32 strain, in which channelrhodopsin-2 fused to enhanced yellow fluorescence protein (ChR2-EYFP) was conditionally expressed in all types of primary somatosensory neurons (advillincre/ChR2+/+). Immunofluorescence and transdermal photostimulation on the hindpaws were used to verify the transgenic mice. Optical stimulation to evoke pain-like paw withdrawal latency was used to assess the analgesic effects of a series of drugs. Injury- and pain-related molecular biomarkers were investigated with immunohistofluorescence. We found that the expression of ChR2-EYFP was observed in many primary afferents of paw skin and sciatic nerves and in primary sensory neurons and laminae I and II of the spinal dorsal horns in advillincre/ChR2+/+ mice. Transdermal blue light stimulation of the transgenic mouse hindpaw evoked nocifensive paw withdrawal behavior. Treatment with gabapentin, some channel blockers, and local anesthetics, but not opioids or COX-1/2 inhibitors, prolonged the paw withdrawal latency in the transgenic mice. The analgesic effect of gabapentin was also verified by the decreased expression of injury- and pain-related molecular biomarkers. These optogenetic mice provide a promising model for assessing the therapeutic efficacy of analgesics in neuropathic pain.

[Screening of the Optimum Medium for Rat Mesenchymal Stem Cells].

OBJECTIVE: To investigate the effect of three different cell culture mediums, DMEM-LG, α-MEM and DMEM/F12, on the growth of rat bone marrow mesenchymal stem cells (BMSCs) in vitro, and so that to screen out the most suitable medium for in vitro culturing the rat BMSCs. METHODS: BMSCS were isolated from the femur and tibia of SD rats by whole bone marrow differential adherence method. The isolated cells were then cultured with three culture mediums, DMEM-LG, α-MEM and DMEM/F12. The rat BMSCs morphology, adhesion, proliferation, the time of passage and the number the colony at day 14 in three mediums respectively were observed with inverted phase contrast microscopy and compared. Flow cytometry was used to identify and observe the effects of different mediums on the surface antigen expression of rats BMSCs. RESULTS: Compared with the other two groups of media, BMSCs cultured in DMEM-LG had shorter colony formation time, shorter first passage time, more clone formation (14±2) and showed uniform morphology and the highest attachment efficiency (47.0±2.8)%. Meanwhile, BMSCs cultured with DMEM-LG entered logarithmic growth phase after only 4 days of culturing and showed the highest average specific growth rate and the largest average number of propagations per unit time. The total number of cells reached about (2.2-2.7)×105 mL-1 within three days. The cells cultured with 3 mediums were all identified as rat BMSCs, and the expression of surface antigen in BMSCs was not significantly affected by different media. CONCLUSION: DMEM-LG is more suitable for proliferation of rat BMSCs in vitro.

[Research on Temporal and Spatial Evolution of Reheating Double-Pulse Laser-Induced Plasma].

In order to investigate the emission enhancement mechanisms of reheating Double Pulse Laser-Induced Breakdown Spectroscopy (DP-LIBS), single pulse LIBS (SP-LIBS) and reheating DP-LIBS were carried out on an alloy steel sample respectively. The plasma emission was collected by an Echelle spectrometer with high resolution, while the plasma structure was monitored via fast-photography. The temporal and spatial evolutio ns of the plasma generated by SP-LIBS and reheating DP-LIBS were being studied. It is found that the plasma temperature in reheating DP-LIBS was higher than that of SP-LIBS, and there was a turning point for the decay rate of plasma temperature in reheating DP-LIBS when the delay time was equal to the interpulse time of DP-LIBS. Moreover, the inte nsity of the plasma image was increased by reheating DP-LIBS, and the height and width of the central region of the plasma were increased about 23.5% and 15.1% respectively. The results of spatial distribution showed that the intensity of Fe II and N I lines in the plasma were obviously enhanced by reheating DP-LIBS when the distance from the sample surface was larger than 0.6 mm. While the intensity enhancement for Fe I lines were little, even in some positio ns the intensity of Fe I lines decreased. The plasma temperature of double-pulse configuration was about 2 000 K higher than that of SP-LIBS, and a larger hot region in the plasma was generated. It is evidenced that the emission enhancement mechanisms in reheating DP-LIBS is that the second laser pulse re-excited the plasma induced by the first laser pulse, and the higher plasma temperature resulted from the re-exciting process.

[Research on algorithm for self-absorption correction based on multi-particles LIBS spectra].

In order to overcome the influence of self-absorption on quantitative analysis, the optimizing process of very fast simulated annealing algorithm was studied. According to basic theory of plasma emission spectrum, a new algorithm for self-absorpton correction based on multi-particles spectra was proposed, and the algorithm flowchart was given. With the self-absorption correction algorithm mentioned above, the spectra of refining slag and blast furnace slag were corrected. The effect of self-ab sorption correction on the quantitative analysis results was analyzed based on calibration free method. Comparison of Boltzmann plots before and after self-absorption correction indicated that the plasma temperatures calculated with spectra after self-absorption correction tended to be uniform, and remained stable around 11,600 K. The Boltzmann plots constructed with plasma spectra of the same particle after self-absorption correction indicated that the intercepts were almost the same except for one group data. With calibration free method and spectra after self-absorption correction, the contents of components in slag were analyzed. For refining slag, quantitative analysis precision of MgO was low. If ignoring the existence of MgO, the relative errors of quantitative analysis results of CaO, Al2 O3 and SiOs were much smaller. For blast furnace slag in which the content of MgO was 8.49%, the relative error of quantitative analysis result of Al2 O3 was 2.38%, which was the smallest. And the relative error of quantitative analysis result of MgO was 28.27%, which was still the biggest.

[A multivariate nonlinear model for quantitative analysis in laser-induced breakdown spectroscopy].

Most quantitative models used in laser-induced breakdown spectroscopy (LIBS) are based on the hypothesis that laser-induced plasma approaches the state of local thermal equilibrium (LTE). However, the local equilibrium is possible only at a specific time segment during the evolution. As the populations of each energy level does not follow Boltzmann distribution in non-LTE condition, those quantitative models using single spectral line would be inaccurate. A multivariate nonlinear model, in which the LTE is not required, was proposed in this article to reduce the signal fluctuation and improve the accuracy of quantitative analysis. This multivariate nonlinear model was compared with the internal calibration model which is based on the LTE condition. The content of Mn in steel samples was determined by using the two models, respectively. A minor error and a minor relative standard deviation (RSD) were observed in multivariate nonlinear model. This result demonstrates that multivariate nonlinear model can improve measurement accuracy and repeatability.

[Quantitative analysis of slag by calibration-free laser-induced breakdown spectroscopy].

Calibration-free laser induced breakdown spectroscopy (CF-LIBS) was employed for the quantitative analysis of slag. Nd:YAG laser ablation was performed in air. The laser-induced plasma emission was measured by an Echelle spectrometer equipped with an ICCD. The plasma temperature and electron number density were determined from Boltzmann plots and a Ca I line width, respectively. The assumption of local thermal equilibrium was validated by the Ne criterion. The concentration of oxides was obtained from the concentration of elements by using stoichiometric relation. The calculated oxide concentrations were compared with those obtained by XRF. The relative errors of major elements were less than 15%. The results indicate that this method can be employed for the analysis of major elements in multi-component complex materials without certified reference.