Development of a liquid chromatography with mass spectrometry method for the determination of gelsemine in rat plasma and tissue: Application to a pharmacokinetic and tissue distribution study

Gelsemine from Gelsemium elegans Benth is a potential anesthetic and analgesic agent with no physical dependence and opiate addiction. This study was aimed at developing an ultrafast liquid chromatography coupled to tandem mass spectrometry method to quantify gelsemine in rat plasma and tissues. Plasma and tissues were processed with acetonitrile precipitation, and dendrobine was chosen as the internal standard. Sample separation was performed on an ACQUITY HSS T3 column. The mobile phase consisted of acetonitrile and 0.1% formic acid aqueous solution. Multiple reactions monitoring mode was utilized to detect the compounds of interest. The mass spectrometer was operated in the positive ion mode for detection. The MS/MS ion transitions monitored were m/z 323.2→70.5 for gelsemine and 264.2→108.05 for dendrobine, respectively. The calibration curves were linear over the range of 1–500 ng/mL in all biological matrices. The lower limit of quantification for rats plasma and tissues was 1.0 ng/mL. The values for inter‐ and intraday precision and accuracy were well within the ranges acceptable (< 15%). It was successfully applied to the pharmacokinetic and tissue distribution studies of gelsemine after intravenous doses of 5, 2, and 0.5 mg/kg in rats. These data of gelsemine would be useful for clinical application and further development.     

大白鼠抑郁症模型的MRI方法研究

用核磁共振成象方法研究对比了抑郁症大白鼠与对照组正常大白鼠脑部的核磁共振信号强度的差别.从大白鼠大脑的核磁共振图象出发,经过数据分析,发现抑郁症大白鼠多个脑组织的核磁共振信号相对强度普遍比正常大白鼠高.另外,模型组大白鼠大脑的脑裂比对照组大白鼠的脑裂更深且更宽.     

Tracking of transplanted mesenchymal stem cells labeled with fluorescent magnetic nanoparticle in liver cirrhosis rat model with 3-T MRI

In vivo visualization of transplanted stem cells with noninvasive technique is essential for the monitoring of cell implantation, homing and differentiation. At present, superparamagnetic iron oxide (SPIO) is most commonly used for cell labeling. However, stem cells lack phagocytic capacity and transfection agent is required for sufficient internalization of SPIO for cellular imaging. However, the potential hazards of transfection agents are not fully investigated. Instead of SPIO, we used commercially available new tagging material, fluorescent magnetic nanoparticle (MNP) containing rhodamine B isothiocyanate within a silica shell (Biterials, Seoul, Korea). This tagging material does not require transfection agents for the cell labeling. In addition to that, the core of this MNP is composed of ferrite and the inner portion of silica shell contains fluorescent materials, therefore, it has both magnetic and optical features. This study was designed to track intrasplenically injected bone marrow mesenchymal stem cells (MSCs) labeled with fluorescent MNP in liver cirrhosis rat model with 3-T magnetic resonance equipment. We compared magnetic resonance imaging (MRI) of livers in rats which were injected with non-labeled stem cells or labeled stem cells with MNP or SPIO. We found that the respective liver-to-muscle contrast-to-noise ratios at 3 and 5 h after MNP or SPIO-labeled stem cell injection was significantly lower than that of pre-injection and non-labeled group. There was no significant difference between MNP-labeled group and SPIO-labeled group. We can effectively detect intrasplenically injected MNP-labeled MSCs in an experimental rat model of liver cirrhosis with 3-T MRI.     

White matter maturation in the brains of Long Evans shaker myelin mutant rats by ex-vivo QSI and DTI

The brains of Long Evans shaker (les) rats, a model of dysmyelination, and their age- matched controls were studied by ex-vivo q-space diffusion imaging (QSI) and diffusion tensor imaging (DTI). The QSI and DTI indices were computed from the same acquisition. The les and the control brains were studied at different stages of maturation and disease progression. The mean displacement, the probability for zero displacement and kurtosis were computed from QSI data while the fractional anisotropy (FA) and the eigenvalues were computed from DTI. It was found that all QSI indices detect the les pathology, at all stages of maturation, while only some of the DTI indices could detect the les pathology. The QSI mean displacement was larger in the les group as compared with their age-matched controls while the probability for zero displacement and the kurtosis were both lower all indicating higher degree of restriction in the control brains. Since all the DTI eigenvalues were higher in the les brains as compared to controls, the less efficient DTI measure for discerning the les pathology was found to be the FA. Clearly, the most sensitive DTI parameter to the les pathology is λ₃, i.e. the minimal diffusivity. Since the QSI and DTI data were obtained from the same acquisition, despite the somewhat higher SNR of the QSI data compared to the DTI data, it seems that the higher diagnostic capacity of the QSI data in this experimental model of dysmyelination, originates mainly from the higher diffusing weighting of the QSI data.     

Natural abundance (17)O NMR spectroscopy of rat brain in vivo

Oxygen is an abundant element that is present in almost all biologically relevant molecules. NMR observation of oxygen has been relatively limited since the NMR-active isotope, oxygen-17, is only present at a 0.037% natural abundance. Furthermore, as a spin 5/2 nucleus oxygen-17 has a moderately strong quadrupole moment which leads to fairly broad resonances (T(2)=1-4 ms). However, the similarly short T(1) relaxation constants allow substantial signal averaging, whereas the large chemical shift range (>300 ppm) improves the spectral resolution of (17)O NMR. Here it is shown that high-quality, natural abundance (17)O NMR spectra can be obtained from rat brain in vivo at 11.74 T. The chemical shifts and line widths of more than 20 oxygen-containing metabolites are established and the sensitivity and potential for (17)O-enriched NMR studies are estimated.     

Snapshot gradient-recalled echo-planar images of rat brains at long echo time at 9.4 T

With improved B 0 homogeneity along with satisfactory gradient performance at high magnetic fields, snapshot gradient-recalled echo-planar imaging (GRE-EPI) would perform at long echo times (TEs) on the order of T2*, which intrinsically allows obtaining strongly T2*-weighted images with embedded substantial anatomical details in ultrashort time. The aim of this study was to investigate the feasibility and quality of long TE snapshot GRE-EPI images of rat brain at 9.4 T. When compensating for B 0 inhomogeneities, especially second-order shim terms, a 200 x 200 microm2 in-plane resolution image was reproducibly obtained at long TE (>25 ms). The resulting coronal images at 30 ms had diminished geometric distortions and, thus, embedded substantial anatomical details. Concurrently with the very consistent stability, such GRE-EPI images should permit to resolve functional data not only with high specificity but also with substantial anatomical details, therefore allowing coregistration of the acquired functional data on the same image data set.     

Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L⁻¹ (n = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.     

In vivo diffusion tensor imaging of thoracic and cervical rat spinal cord at 7 T

In vivo diffusion tensor imaging (DTI) of rat cervical and thoracic spinal cord was performed using a three-element phased array coil at 7 T. The magnetic field was shimmed over the spinal cord in real time using an in-house developed automatic algorithm. Echo planar imaging (EPI)-based diffusion-weighted images (DWIs) were acquired with 21 gradient encoding directions. The DWIs were tensor encoded, and diffusion tensor metrics, fractional anisotropy (FA), mean diffusivity (MD), longitudinal diffusivity (λ₀) and transverse diffusivity (λ) were determined for both white matter (WM) and gray matter (GM). The results on six normal rats indicated no significant differences in the diffusion tensor metrics between thoracic and cervical regions. However, the DTI-derived metrics in cervical spinal cord from our study are somewhat different from the published results in rats. The possible reasons for these differences are suggested.     

Neuronal projections from ventral tegmental area to forebrain structures in rat studied by manganese-enhanced magnetic resonance imaging

Manganese-enhanced magnetic resonance imaging (MEMRI) has been widely applied to trace neuronal tracts and to monitor morphological and functional responses of specific brain circuits to changes in physiological and/or environmental conditions. In this study, we traced the efferent axonal projections from ventral tegmental area (VTA) to forebrain structures, an integrating part of the reward circuit implicated in drug addiction, in rats using MEMRI. Urethane- and chloral hydrate-anesthetized rats received injection of 100 nl of 200 mM MnCl(2) solution into the right VTA. Mn(2+)-induced signal enhancements were monitored 24 h after injection. The dose of MnCl(2) injection was shown, by histological evaluation, to have minor toxic effects to the neurons in/near the injection site. Dynamic Mn(2+)-induced signal intensity changes in urethane-anesthetized rats during a 24-h period were fit to a sigmoidal function to obtain parameters slope and t(50), which describe the dynamics of apparent Mn(2+)accumulation. The results showed that most of the forebrain structures known to receive neuronal projections from the VTA, including prefrontal cortex, nucleus accumbens, globus pallidus and caudate putaman, were enhanced at 24 h after injection of MnCl(2) into the ipsilateral VTA, and anesthesia seemed have little effects on the amount of Mn(2+)being transported from the VTA to these structures.     

Intravascular ultrasound allows a hyperplasia diagnosis in vivo in rat as accurate as histomorphometry

Objective and motivation

Intravascular ultrasound (IVUS) allows in vivo invasive intra-luminal real-time examination of the arterial wall structure. In this study, we aimed to validate for the first time the in vivo IVUS performing as a diagnostic tool by comparison to the well-established histomorphometry approach, in the largely used rat model of carotid angioplasty model that mimics the angioplasty procedure in humans.

Methods

Atherosclerotic lesions were allowed to develop during four weeks after balloon catheter inflation of the left carotid artery, whereas the intact right carotid artery was used as control. Four weeks after injury, a Boston Scientific 40 MHz device to perform IVUS exams in vivo on both carotid arteries. Then, both carotid arteries were examined in vitro by histomorphometry and correlation between IVUS and histomorphometric parameters (plaque plus media cross-sectional areas [CSA] and eccentricity index) were researched.

Results

After ANOVA analysis, comparative statistical analysis showed significant correlations between IVUS and histomorphometry when examining the intact right carotid artery (r = 0.662 with p < 0.003 for plaque plus media CSA; r = 0.774 with p < 0.002 for eccentricity index), but also when exploring the injured left carotid artery (r = 0.805 with p < 0.0001 for plaque plus media CSA; r = 0.775 with p < 0.002 for eccentricity index).

Conclusions and outcome

We report here for the first time the ability of IVUS to study therapeutic vascular effects in vivo in alive rats. This result is of major importance since it will allow this device to be used for restenosis drug testing in rat model of carotid angioplasty.