Photoacoustic microscopy

Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (∼1 mm in soft tissue). With its excellent scalability, PAM can provide high‐resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering‐based confocal microscopy and optical coherence tomography, PAM provides absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence‐based methods, such as wide‐field, confocal, and multi‐photon microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state‐of‐the‐art developments in PAM, this Review discusses the key features of PAM implementations and their applications in biomedical studies.     

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.     

Cell protection from Ca2+-overloading by bioactive molecules extracted from olive pomace

We are reporting in the present study that molecules extracted from olive pomace prevent cell death induced by Ca²⁺-overloading in different cell types. Exposure of cells to these molecules counteracts the Ca²⁺-induced cell damages by reducing the activation of the Ca²⁺-dependent protease calpain, acting possibly through the modification of the permeability to Ca²⁺ of the plasma membrane. The purification step by RP-HPLC suggests that effective compound(s), differing from the main biophenols known to be present in the olive pomace extract, could be responsible for this effect. Our observations suggest that bioactive molecules present in the olive pomace could be potential candidates for therapeutic applications in pathologies characterised by alterations of intracellular Ca²⁺ homeostasis.     

Agave fructans and oligofructose decrease oxidative stress in brain regions involved in learning and memory of overweight mice

Obesity is currently a public health problem worldwide. Recently, non-reducing carbohydrates, that include β(2→1) and β(2→6) linkages in their structure, have been of particular interest in the field of obesity because they are involved in lipid metabolism. Some of these are agave fructans (AF) and oligofructose (OF). In this study, we evaluated both AF and OF on oxidative stress (OS) markers in the brain of overweight mice (OM). AF and OF decreased TBARS levels and carbonyls at different levels in hippocampus (HP), frontal cortex (FC) and cerebellum (CB) of OM. The results indicated that fructans may have anti-oxidative potential and can be used as an alternative treatment for the prevention of the consequences of this pathology.     

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.     

Controlled positioning of microbubbles and induced cavitation using a dual-frequency transducer and microfiber adhesion techniques

We report a study on two methods that enable spatial control and induced cavitation on targeted microbubbles (MBs). Cavitation is known to be present in many situations throughout nature. This phenomena has been proven to have the energy to erode alloys, like steel, in propellers and turbines. It is recently theorized that cavitation occurs inside the skull during a traumatic-brain injury (TBI) situation. Controlled cavitation methods could help better understand TBIs and explain how neurons respond at moments of trauma. Both of our approaches involve an ultrasonic transducer and bio-compatible Polycaprolactone (PCL) microfibers. These methods are reproducible as well as affordable, providing more control and efficiency compared to previous techniques found in literature. We specifically model three-dimensional spatial control of individual MBs using a 1.6 MHz transducer. Using a 100 kHz transducer, we also illustrate induced cavitation on an individual MB that is adhered to the surface of a PCL microfiber. The goal of future studies will involve characterization of neuronal response to cavitation and seek to unmask its linkage with TBIs.     

Studies on neurosteroids XXII. Liquid chromatography-tandem mass spectrometric method for profiling rat brain 3-oxo-4-ene-neuroactive steroids

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous determination of five 3-oxo-4-ene-neuroactive steroids, i.e. androstenedione, testosterone (T), progesterone (PROG), 20alpha-dihydroprogesterone and 20beta-dihydroprogesterone, in rat brain has been developed and validated. The brain steroids were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges and subjected to LC-ESI-MS/MS. The method does not require derivatization. Deuterium-labeled T and PROG were used as the internal standards, and quantification was based on the selected reaction monitoring mode. This method allowed the reproducible and accurate quantification of the brain neuroactive steroids using 100 mg of tissue; the intra- and inter-assay relative standard deviations were below 4.7 and 4.3%, respectively, and the accuracy values were 97.6-103.2% for all the steroids. The limits of quantitation were 0.1 ng/g tissue for all the steroids. The application of this developed method for the analysis of changes in the brain neuroactive steroid levels by immobilization stress is also presented.     

Stereoselective determination of demethyl- and didemethyl-citalopram in rat plasma and brain tissue by liquid chromatography with fluorescence detection using precolumn derivatization

A rapid and sensitive HPLC enantioselective method with fluorescence detection was developed to determine (-)-(R) and (+)-(S) enantiomers of the metabolites of citalopram, demethyl- and didemethyl-citalopram in plasma and brain tissue. This assay involves pre-column chiral derivatization with (-)-(R)-1-(1-naphthyl)ethyl isocyanate followed by separation on a normal-phase silica column. The developed liquid-liquid extraction procedure permits quantitative determination of analytes with recoveries ranged between 81 and 88% with intra- and inter-day relative standard deviations less than 10.5%. Linearity was obtained over the concentration range 5-1000 ng/mL and 100-10,000 ng/g for spiked drug-free plasma and brain tissue, respectively, with detection limits lower than 2.1 ng/mL and 42.8 ng/g.     

大鼠脑组织含磷代谢物随年龄变化的NMR研究

随着人口老龄化的不断加剧,衰老相关疾病已成为全球关注的问题.神经系统功能的退行性改变居于衰老相关疾病的首位,其机制尚不明了.该文旨在通过核磁共振磷谱(~(31)P NMR)检测不同月龄大鼠脑组织中小分子物质的改变,明确能量相关的含磷化合物随大鼠年龄增长的变化,从而揭示脑组织中年龄相关代谢物的变化规律.该研究萃取了不同年龄(幼年、中年及老年)Sprague-Dawley(SD)大鼠脑组织中的小分子物质,进行了~(31)P NMR检测分析.结果显示:老年大鼠脑组织中能量代谢相关物质——磷酸肌酸的含量显著升高,而磷脂酰胆碱及磷酸肌醇含量显著降低,并且出现了磷酸化葡萄糖.这一发现有助于理解衰老相关的脑结构和功能下调,为神经退行性疾病的发生提供依据.     

Methodological aspects of in vitro sensing of L-glutamate in acute brain slices

L-Glutamate is a major amino acid neurotransmitter in the central neuronal system of the mammalian brain and plays a vital role in brain development, synaptic plasticity, neurotoxicity, and neuropathological disorders. Despite technical limitations, progress is being made in sensing L-glutamate in vivo and in vitro. Sophisticated microsensors with the necessary spatial and temporal resolution have recently been emerging, which enable us to discern regional distribution, concentration levels, and temporal changes of L-glutamate in acute brain slices. The L-glutamate sensors for in vitro sensing have different structures and sizes, such as glass capillary-based enzyme sensors, polymer-coated enzyme sensors, and patch sensors based on natural sensing probes. The concentration of L-glutamate released in brain slices by chemical stimulation is markedly dependent on neuronal regions, types of stimulation, and sensing methods. Real- and long-time monitoring of L-glutamate in acute hippocampal slices is beginning to shed light on L-glutamate release related to the molecular mechanisms of long-term potentiation. Progress is also being made toward the visualization of L-glutamate release in acute hippocampal slices. The methodological aspects of in vitro sensing of L-glutamate are discussed.