Lingyan Shi

Quantification of transient increase of the blood–brain barrier permeability to macromolecules by optimized focused ultrasound combined with microbubbles

Radioimmunotherapy using a radiolabeled monoclonal antibody that targets tumor cells has been shown to be efficient for the treatment of many malignant cancers, with reduced side effects. However, the blood–brain barrier (BBB) inhibits the transport of intravenous antibodies to tumors in the brain. Recent studies have demonstrated that focused ultrasound (FUS) combined with microbubbles (MBs) is a promising method to transiently disrupt the BBB for the drug delivery to the central nervous system. To find the optimal FUS and MBs that can induce reversible increase in the BBB permeability, we employed minimally invasive multiphoton microscopy to quantify the BBB permeability to dextran-155 kDa with similar molecular weight to an antibody by applying different doses of FUS in the presence of MBs with an optimal size and concentration. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a micro-grinder. About 5 minutes after applying the FUS on the thinned skull in the presence of MBs for 1 minute, TRITC (tetramethylrhodamine isothiocyanate)-dextran-155 kDa in 1% bovine serum albumin in mammalian Ringer’s solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump. Simultaneously, the temporal images were collected from the brain parenchyma ~100–200 μm below the pia mater. Permeability was determined from the rate of tissue solute accumulation around individual microvessels. After several trials, we found the optimal dose of FUS. At the optimal dose, permeability increased by ~14-fold after 5 minutes post-FUS, and permeability returned to the control level after 25 minutes. FUS without MBs or MBs injected without FUS did not change the permeability. Our method provides an accurate in vivo assessment for the transient BBB permeability change under the treatment of FUS. The optimal FUS dose found for the reversible BBB permeability increase without BBB disruption is reliable and can be applied to future clinical trials.

Deep Imaging in Tissue and Biomedical Materials : Using Linear and Nonlinear Optical Methods

biomedical imaging—a field that has been transformed by a variety of technical innovations in recent years. Editors Shi and Alfano have secured contributions from top names in the field, for an extensive compilation that comprehensively details the new state of the art, including forefront advances and developments. Fully covering theory, methods and applications, this lavishly illustrated book is destined to become a reference classic.” Prof. David L. Andrews University of East Anglia, UK

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

Alzheimer's disease: evaluation using label-free, stainless, fluorescence of tryptophan metabolites and the kynurenine pathway

Under stress conditions, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin 6 and interferon gamma are released. It is known that these cytokines stimulate indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), which increase tryptophan metabolism through the kynurenine pathway, and that this can cause increased production of neurotoxic compounds. Brain tissues from Alzheimer’s disease patients and agematched controls were investigated using label-free fluorescence spectroscopy. Tryptophan (exc. 280/ em. 340 nm) and its metabolites (N-formyl-L-kynurenine (exc. 325/em. 434 nm), kynurenine (exc. 365/em. 480 nm) and kynurenic acid (exc. 330/em. 390 nm)) have distinct spectral profiles. Preliminary results show a difference in the optical signatures in three important areas of the brain (hippocampus, BA 9, BA 17) between patients with Alzheimer’s disease and agedmatched controls (normal), and a marked relative increase in tryptophan in the Alzheimer’s patients. Thus determinations of tryptophan to tryptophan metabolite ratios could potentially be used to measure IDO and TDO activity and the degree of inflammation in the brain. This label-free optical technique may be useful in the study of Alzheimer’s and other neurodegenerative diseases.

Resonance Raman imaging for detecting and monitoring molecular pathological changes in human brain tumors related to Warburg effect

The goal of the research is to determine the prognostic molecular pathological changes in components and composition, for human brain glioma gradings in comparison with normal tissues in three-dimensional Raman imaging profiles by visible Resonance Raman (VRR) imaging. VRR images from twenty-five specimens including three healthy tissues, one normal control, and twenty-one glioma tissues of grades II, II-III and III-IV with histology examination were measured and investigated using WITec300R confocal micro Raman imaging system with laser excitation of 532nm. Two-dimensional RR spectral mappings performed in 20μm x 20μm generated 400 images which integrated the intensity of the specific biochemical bonds as the third dimension. The three-dimension (3D) map demonstrated the spatial distributions of three selected sets of RR spectra of molecular biomarkers, and revealed significant differences in the spectra between normal and glioma tissues of different grades due to the composition changes in key molimageecules. These RR molecular spectral fingerprints have displayed: a clear enhancement of RR vibrational modes at 1129-1131cm-1 and 2934cm-1 which are supposed to be arising from lipoproteins; evident decreased RR vibrational modes at 1442cm-1 and 2854cm-1 which are from saturated fatty acids bonds in all-grades of glioma brain tissues compared with normal tissues; and the enhanced RR spectral modes of 1129 cm-1 and 2938cm-1 which suggest contribution from lactate. These findings may provide a novel proof for anaerobic glycolysis metabolic process in brain glioma cancer tissues that has been explained by Warburg effects.

Statistical analysis and machine learning algorithms for optical biopsy

Analyzing spectral or imaging data collected with various optical biopsy methods is often times difficult due to the complexity of the biological basis. Robust methods that can utilize the spectral or imaging data and detect the characteristic spectral or spatial signatures for different types of tissue is challenging but highly desired. In this study, we used various machine learning algorithms to analyze a spectral dataset acquired from human skin normal and cancerous tissue samples using resonance Raman spectroscopy with 532nm excitation. The algorithms including principal component analysis, nonnegative matrix factorization, and autoencoder artificial neural network are used to reduce dimension of the dataset and detect features. A support vector machine with a linear kernel is used to classify the normal tissue and cancerous tissue samples. The efficacies of the methods are compared.

Resonance Raman and fluorescence spectroscopy to evaluate increased brain kynurenine pathway activity in samples from patients with neurodegenerative disease

Resonance Raman and fluorescence spectroscopy were used to assess increased kynurenine pathway activity in brain samples from Alzheimer’s patients and age-matched controls. Increased activity was seen in areas of the brain involved in Alzheimer’s disease.

An optical window for deep brain imaging

One of the major goals in neuroscience is to image the structure of the brain at cellular resolution. However, achieving deep brain tissue imaging has posed a significant challenge because of technical limitations in accessing wavelengths beyond 950nm. Recently, the availability of new technologies, such as suitable near-infrared (NIR) detectors and femtosecond laser sources, offer great potential for deep brain imaging. Now, we have discovered a ‘golden window’ that uses NIR light at wavelengths of 1600–1870nm, which offers the optimal transmittance for deep brain imaging.1, 2 The brain is a unique tissue, made up of a massive network of neural cells. It contains mostly water, and has twice as much lipid, and less than half the protein of muscle tissue. All these components make brain imaging a challenge. Although the widely used technique of magnetic resonance imaging (MRI) is not limited by depth, it can only image brain tissue at the millimeter scale. Optical imaging is still the only applicable method to study neural tissues with resolution at the micrometer or sub-micrometer scale. When a light pulse propagates in tissue media it divides into separate components: ballistic, snake, and diffusive.3, 4 Traditional multiphoton microscopy enables deep-tissue imaging by using only the ballistic component of light to excite fluorescence at the visible wavelength. However, when using visible light for brain imaging, the penetration depth is limited by the high degree of light scattering (which blurs images) and absorption (which reduces the number of available photons) in the tissue. The application of NIR at wavelengths 650–950nm (optical window I) reduces the absorption and scattering, and increases the penetration depth. Using new tools, such as NIR femtosecond lasers and photodetectors that are based on indium gallium Figure 1. Transmittance T (%) (a) and absorbance (b) in rat brain tissue in optical windows I, II, III, and IV. Brain tissue thicknesses were 50, 100, 150, and 200 m.

Deep Brain Imaging using the Near-Infrared Golden Optical Window Wavelengths

This study applies optical window (III, 1,600-1,870 nm) for deep brain imaging. In vivo experiment was conducted on mice with skull thinning and skull intact by using a homemade 1620nm multiphoton microscopy.