Yuri Kuznetsov

Label free in vivo laser speckle imaging of blood and lymph vessels

The peripheral lymphatic vascular system is a part of the immune body system comprising a complex network of lymph vessels and nodes that are flowing lymph toward the heart. Traditionally the imaging of lymphatic vessels is based on the conventional imaging modalities utilizing contrast fluorescence materials. Given the important role of the lymphatic system there is a critical need for the development of noninvasive imaging technologies for functional quantitative diagnosis of the lymph vessels and lymph flow without using foreign chemicals. We report a label free methodology for noninvasive in vivo imaging of blood and lymph vessels, using long-exposure laser speckle imaging approach. This approach entails great promise in the noninvasive studies of tissues blood and lymph vessels distribution in vivo.

Transcranial optical vascular imaging (TOVI) of cortical hemodynamics in mouse brain

In vivo imaging of cerebral vasculature and blood flow provides highly valuable information for clinicians as well as researchers. Nevertheless, currently available methods are complex, time-consuming and expensive. Here, we present a novel, minimally invasive method for vascular imaging through the sufficiently transparent intact skull of young mice. Our method combines laser speckle and fluorescent imaging with dynamic color mapping and image fusion. Quickly generated wide-field images present clear visual information on blood flow and perfusion in the cerebral cortex and meninges. The ability of the method to visualize hemodynamic changes is demonstrated by induced occlusion of the middle cerebral artery. The compact and easily operated system comprises of several pieces of standard and affordable laboratory equipment. This simple, robust and inexpensive method may become an important tool for assessment of brain hemodynamics in preclinical studies.

A simple approach for non-invasive transcranial optical vascular imaging (nTOVI).

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non-invasive optical-based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non-invasive optical-based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro-imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models. An example of 1 × 1.5 cm color-coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.

Ear swelling test by using laser speckle imaging with a long exposure time

Abstract. Laser speckle imaging with long exposure time has been applied noninvasively to visualize the immediate reaction of cutaneous vessels in mice in response to a known primary irritant and potential allergen—methyl salicylate. The compound has been used topically on the surface of the pinna and the reaction of the vascular network was examined. We demonstrate that irritant-induced acute vascular reaction can be effectively and accurately detected by laser speckle imaging technique. The current approach holds a great promise for application in routine screening of the cutaneous vascular response induced by contact agents, screenings of mouse ear swelling test, and testing the allergenic potential of new synthetic materials and healthcare pharmaceutical products.

Targeted Delivery of Adipose Derived Stem Cells into a Transplant by Direct Intra-Arterial Administration

Objective: Mesenchymal Stem Cells (MSCs) are adult multipotent cells that possess regenerative and immunosuppressant properties. Homing of MSCs to target organs remains a major challenge as intravenous delivery results in intravascular entrapment of most MSCs in vascularized organs. Intra-Arterial (IA) administration of MSCs to arteries feeding a specific organ improved the delivery of cells to these organs but often resulted in vessels obstruction. To improve targeting of MSCs into a transplant we designed a novel method for IA delivery of MSCs during the transplantation procedure. This study was aimed at evaluating the safety and efficacy of this method. Methods: A syngeneic groin free flap between Lewis rats was performed in all experiment groups. Treatment groups included 3 groups (n ≥ 7) in which 1 × 106, 0.5 × 106 or 0.05 × 106 adipose derived MSCs (ASCs) were administered via a femoral artery branch prior to the final reperfusion of the flap. In vivo real time fluorescence imaging and intravital microscopy were used to define ASCs IA movement after transplantation. Results: High concentrations of ASCs per injection resulted in poor flap survival rates (14.3%) due to flap necrosis. At 0.05 × 106 ASCs, increased long-term flap viability rates (85%) were observed. Whole-body imaging of fluorescently labeled ASCs demonstrated significant targeting of cells into the flap even at such a low cell quantity. ASCs were detected in proximity to small blood vessels within the viable flap. Conclusions: Local IA administration of ASCs into a vascularized transplant/flap is feasible and allows high local cell concentrations with minimal cell dosing.

Multimodal diagnostic approach for functional imaging of tumor vascular network and blood microcirculation

We present a multi-modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy and Dynamic Light Scattering for in vivo functional imaging of tumor vascular network and blood microcirculation. Fluorescence Intravital Microscopy is used for imaging of tumor and tumor surroundings, whereas Dynamic Light Scattering is applied for imaging of vascular network and blood microcirculation. The obtained results demonstrate that presented multi-modal imaging approach has a great potential in vascular biology and can significantly expand the capabilities of tumor angiogenesis studies and notably contribute to the development of cancer treatment.

Dual role of E-cadherin in the regulation of invasive collective migration of mammary carcinoma cells

In this article, we explore a non-canonical form of collective cell migration, displayed by the metastatic murine mammary carcinoma cell line 4T1. We show here that in sparsely plated 4T1 cells, E-cadherin levels are moderately reduced (~50%), leading to the development of collective migration, whereby cells translocate in loose clusters, interconnected by thin membrane tethers. Knocking down E-cadherin blocked tether formation in these cells, leading to enhancement of migration rate and, at the same time, to suppression of lung metastases formation in vivo, and inhibition of infiltration into fibroblast monolayers ex vivo. These findings suggest that the moderate E-cadherin levels present in wild-type 4T1 cells play a key role in promoting cancer invasion and metastasis.

Regulatory Forum Opinion Piece∗: Imaging Applications in Toxicologic Pathology-Recommendations for Use in Regulated Nonclinical Toxicity Studies

Available imaging systems for use in preclinical toxicology studies increasingly show utility as important tools in the toxicologic pathologist’s armamentarium, permit longitudinal evaluation of functional and morphological changes in tissues, and provide important information such as organ and lesion volume not obtained by conventional toxicology study parameters. Representative examples of practical imaging applications in toxicology research and preclinical studies are presented for ultrasound, positron emission tomography/single-photon emission computed tomography, optical, magnetic resonance imaging, and matrix-assisted laser desorption ionization—imaging mass spectrometry imaging. Some of the challenges for making imaging systems good laboratory practice–compliant for regulatory submission are presented. Use of imaging data on a case-by-case basis as part of safety evaluation in regulatory submissions is encouraged.

Transcranial optical vascular imaging (TOVI) during cardiac arrest (Conference Presentation)

Based on the recent studies the prognosis of patients after cardiac arrest (CA) remains poor. Thus it is extremely important to understand fine mechanisms related to the influence of CA on the brain and Cerebral Blood Flow (CBF) during and after cardiac arrest. Recently our group introduced Transcranial Optical Vascular Imaging (TOVI) approach that combines laser speckle and dynamic fluorescent imaging. TOVI proved to be useful during various preclinical brain research applications. For example it allows imaging of brain blood vessels of a mouse in vivo through the intact cranium. Herein for the first time we present the use of TOVI during cardiac arrest. TOVI possibly could be a useful tool for preclinical studies of CBF during and after CA.

FXYD5 (dysadherin) may mediate metastatic progression through regulation of the β-Na+-K+-ATPase subunit in the 4T1 mouse breast cancer model

FXYD5 is a Na+-K+-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5-specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5-related morphological changes are mediated through its interaction with Na+-K+-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression may downregulate the β1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5-expressing cells may correlate with matrix metalloproteinase 9 secretion and adhesion changes in 4T1 wild-type cells.