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Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx3Cr1+ macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

In last decade functional brain mapping has made significant strides through development of advanced methods, culminating into revolutionary diagnostic imaging and therapeutic applications of neurophotonics. Imaging technologies include intrinsic optical imaging, voltage-sensitive dye, photoacoustic, optical coherence tomography, multi-spectral imaging, UV, yellow light, thermal and near-infrared spectroscopy. Some of these technologies are not only used in animal studies of the model of epileptic seizures but also been in clinical trials. However, translation of such basic science application of brain mapping technologies into clinical setting remains challenging. In this paper we review current advances in the field, along with one clear focus on laser speckle contrast imaging and its application in epilepsy. Our conclusion is that functional brain optical imaging could play a key role in bridging between morphology and functional activity of the brain, and thus contribute to more accurate diagnostics and improved efficacy of the therapy. Coupling brain optical imaging with measurements of disease biomarkers and adding as well as other neuroscience techniques is making early diagnosis more effective and applicable for variable clinical tasks.

Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Signals from the CLL microenvironment promote progression of the disease and induce drug resistance. This phenomenon is largely dependent on direct contact between the malignant B cells and stromal cells. CD84 belongs to the signaling lymphocyte activation molecule family of immunoreceptors, which self-associates, forming an orthogonal homophilic dimer. We therefore hypothesized that CD84 may bridge between CLL cells and their microenvironment, promoting cell survival. Our in vitro results show that CD84 expressed on CLL cells interact with CD84 expressed on cells in their microenvironment, inducing cell survival in both sides. Blocking CD84 in vitro and in vivo disrupt the interaction of CLL cells with their microenvironment, resulting in induced cell death. Thus, our findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

The choroid plexus epithelium within the brain ventricles orchestrates blood‐derived monocyte entry to the central nervous system under injurious conditions, including when the primary injury site is remote from the brain. Here, we hypothesized that the retinal pigment epithelium (RPE) serves a parallel role, as a gateway for monocyte trafficking to the retina following direct or remote injury. We found elevated expression of genes encoding leukocyte trafficking determinants in mouse RPE as a consequence of retinal glutamate intoxication or optic nerve crush (ONC). Blocking VCAM‐1 after ONC interfered with monocyte infiltration into the retina and resulted in a local pro‐inflammatory cytokine bias. Live imaging of the injured eye showed monocyte accumulation first in the RPE, and subsequently in the retina, and peripheral leukocytes formed close contact with the RPE. Our findings further implied that the ocular milieu can confer monocytes a phenotype advantageous for neuroprotection. These results suggest that the eye utilizes a mechanism of crosstalk with the immune system similar to that of the brain, whereby epithelial barriers serve as gateways for leukocyte entry.

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.

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.

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.

We describe the laser speckle contrast method for simultaneous noninvasive imaging of blood and lymphatic vessels of living organisms, based on increasing detector exposure time. In contrast to standard methods of fluorescent angiography, this technique of vascular bed imaging and lymphatic and blood vessel demarcation does not employ toxic fluorescent markers. The method is particularly promising with respect to the physiology of the cardiovascular system under in vivo conditions.

The use of fluorescence imaging of vascular permeability becomes a golden standard for assessing the inflammation process during experimental immune response in vivo. The use of the optical fluorescence imaging provides a very useful and simple tool to reach this purpose. The motivation comes from the necessity of a robust and simple quantification and data presentation of inflammation based on a vascular permeability. Changes of the fluorescent intensity, as a function of time is a widely accepted method to assess the vascular permeability during inflammation related to the immune response. In the present study we propose to bring a new dimension by applying a more sophisticated approach to the analysis of vascular reaction by using a quantitative analysis based on methods derived from astronomical observations, in particular by using a space-time Fourier filtering analysis followed by a polynomial orthogonal modes decomposition. We demonstrate that temporal evolution of the fluorescent intensity observed at certain pixels correlates quantitatively to the blood flow circulation at normal conditions. The approach allows to determine the regions of permeability and monitor both the fast kinetics related to the contrast material distribution in the circulatory system and slow kinetics associated with extravasation of the contrast material. Thus, we introduce a simple and convenient method for fast quantitative visualization of the leakage related to the inflammatory (immune) reaction in vivo.

Noninvasive imaging is a critical part of the study of developing embryos/fetuses, particularly in the context of alterations of gene expression in genetically modified animals. However, in litter-bearing animals, such as mice, the inability to accurately identify individual embryo/fetus in utero is a major obstacle to longitudinal, noninvasive in vivo studies. Arterial spin labeling MRI was adopted here to determine the fetal order along the uterine horns in vivo, based on the specific pattern of dual arterial blood supply within the mouse uterine horns. Blood enters the mouse uterus cranially through the ovarian artery and caudally through the uterine artery. Saturation slices were alternately placed on the maternal heart or on the bifurcation point of the common iliac artery, thereby saturating either downward inflow via the ovarian arteries or upward inflow via the uterine arteries, respectively. Saturation maps provided a unique signature with highly significant correlation between the direction-dependent magnetization transfer and the position of the fetuses/placentas along the uterine horns. The bidirectional arterial spin labeling-MRI method reported here opens possibilities to determine and pursue phenotypic alterations in fetuses and placentas in longitudinal studies of transgenic and knockout mice models, and for studying defects in placental vascular architecture.

We present a multimodal optical diagnostic approach for simultaneous non-invasive in vivo imaging of blood and lymphatic microvessels, utilising a combined use of fluorescence intravital microscopy and a method of dynamic light scattering. This approach makes it possible to renounce the use of fluorescent markers for visualisation of blood vessels and, therefore, significantly (tenfold) reduce the toxicity of the technique and minimise side effects caused by the use of contrast fluorescent markers. We demonstrate that along with the ability to obtain images of lymph and blood microvessels with a high spatial resolution, current multimodal approach allows one to observe in real time permeability of blood vessels. This technique appears to be promising in physiology studies of blood vessels, and especially in the study of peripheral cardiovascular system in vivo.

Objective Microenvironmental interactions of malignant B cells can modulate various in vitro physiological responses, including proliferation, migration, apoptosis, and drug resistance. Disease manifestations of human malignant B-cell variants, isolated based on their differential interactions with fibronectin, were examined in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Materials and Methods Disease manifestations were assessed by pathological examinations and skeletal imaging of NOD/SCID mice injected with malignant B-cell variants. Dissemination patterns were analyzed by whole-body real-time imaging of mice injected with fluorescence-labeled malignant cells. Results Initial dissemination patterns and dynamics of both high (type A) and low (type F)-adherent variants, following intravenous inoculation, were similar. Both cell types reached the spleen and liver within 30 minutes after injection, then increasingly accumulated within the bone marrow. Mice injected with type-A cells developed multiple myeloma–like disease within the bone marrow, with multiple lytic bone lesions. In contrast, type-F cells displayed low tumorigenic capacity in spite of their efficient homing to the bone marrow niche. In addition, type-A cells grew as extramedullary tumors in some of the intravenous-inoculated mice, and formed solid tumors following subcutaneous injection. Both cell variants retained their characteristics surface markers following in vivo outgrowth as tumors, indicating that at least some of their properties are relatively stable. Conclusion Data suggest that the differential tumorigenicity of B-cell adhesive variants is attributable to the capacity of type-A cells to survive and proliferate within the bone marrow, rather than to different initial dissemination of the two cell populations.

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease.

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.

Tumor-associated stroma, in general, and tumor fibroblasts and myofibroblasts, in particular, play a role in tumor progression. We previously reported that myofibroblast infiltration into implanted ovarian carcinoma spheroids marked the exit of tumors from dormancy and that these cells contributed to vascular stabilization in ovarian tumors by expression of angiopoietin-1 and angiopoietin-2. Ex vivo labeling of fibroblasts with either magnetic resonance or optical probes rendered them detectable for in vivo imaging. Thus, magnetic resonance imaging (MRI) follow-up was feasible by biotin-bovine serum albumin-gadolinium diethylenetriaminepentaacetic acid or iron oxide particles, whereas labeling with near-IR and fluorescent vital stains enabled in vivo visualization by near-IR imaging and two-photon microscopy. Using this approach, we show here that prelabeled fibroblasts given i.p. to CD-1 nude mice can be followed in vivo by MRI and optical imaging over several days, revealing their extensive recruitment into the stroma of remote s.c. MLS human epithelial ovarian carcinoma tumors. Two-photon microscopy revealed the alignment of these invading fibroblasts in the outer rim of the tumor, colocalizing with the angiogenic neovasculature. Such angiogenic vessels remained confined to the stroma tracks within the tumor and did not penetrate the tumor nodules. These results provide dynamic evidence for the role of tumor fibroblasts in maintenance of functional tumor vasculature and offer means for image-guided targeting of these abundant stroma cells to the tumor as a possible mechanism for cellular cancer therapy. [Cancer Res 2007;67(19):9180–9]

The ovary is a dynamic organ that undergoes dramatic remodeling throughout the ovulatory cycle. Maturation of the ovarian follicle, release of the oocyte in the course of ovulation as well as formation and degradation of corpus luteum involve tightly controlled remodeling of the extracellular matrix and vasculature. Ovarian tumors, regardless of their tissue of origin, dynamically interact with the ovarian microenvironment. Their activity in the tissue encompasses recruitment of host stroma and immune cells, attachment of tumor cells to mesothelial layer, degradation of the extracellular matrix and tumor cell migration. High-resolution dynamic imaging of such processes is particularly challenging for internal organs. The implementation of a novel imaging window as reported here enabled longitudinal microscopy of ovarian physiology and orthotopic tumor invasion.

The mononuclear phagocyte (MP) system is a body-wide macrophage (MΦ) and dendritic cell (DC) network, which contributes to tissue homeostasis, inflammation, and immune defense. The in vivo origins of MPs remain poorly understood. Here, we use an adoptive precursor cell transfer strategy into MP-depleted mice to establish the in vivo differentiation sequence from a recently identified MΦ/DC-restricted bone marrow (BM) precursor (MDP) via BM and blood intermediates to peripheral MΦs and DCs. We show that MDPs are in vivo precursors of BM and blood monocytes. Interestingly, grafted Gr1high “inflammatory” blood monocytes shuttle back to the BM in the absence of inflammation, convert into Gr1low monocytes, and contribute further to MP generation. The grafted monocytes give rise to DCs in the intestinal lamina propria and lung, but not to conventional CD11chigh DCs in the spleen, which develop during homeostasis from MDPs without a monocytic intermediate.

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis. (Cancer Res 2006; 66(16): 8037-41)

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.

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.

SUMMARY Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the ‘‘alternatively activated’’ anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6c hi CX3CR1 lo ) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6c lo CX3CR1 hi ) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brainventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function.

The bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naive CD8+ T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

We present a multi-modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy (FIM), Dynamic Light Scattering (DLS) and Spectrally Enhanced Microscopy (SEM) modalities for in vivo imaging of tumor vascular network and blood microcirculation. FIM is used for imaging of tumor surroundings and microenvironment, SEM provides information regarding blood vessels topography, whereas DLS is applied for functional imaging of vascular network and blood microcirculation. This complementary combination of the imaging approaches is extremely useful for functional in vivo imaging of blood vasculature and tumor microenvironment. The technique has also 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.

In current report we present synchronized in vivo imaging of tumor vascular network and tumor microenvironment obtained by combined use of Dynamic Light Scattering Imaging, Spectrally Enhanced Microscopy, and Fluorescence Intravital Microscopy. Dynamic Light Scattering Imaging is used for functional imaging of the vascular network and blood microcirculation. Spectrally Enhanced Microscopy provides information regarding blood vessel topography. Fluorescence Intravital Microscopy is used for imaging of tumor microvasculature and tumor microenvironment. These well known modalities have been comprehensively validated in the past and are widely used in various bio-medical applications. As shown here, their combined application has great potential for studies of vascular biology. This multi-modal non-invasive diagnostic technique expands our current capacity to investigate blood microcirculation and tumor angiogenesis in vivo, thereby contributing to the development of cancer research and treatment.

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34 + cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34 + cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in vitro and their recovery in transplanted recipients in vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

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.

After retinal injury in mice, infiltrating monocyte-derived macrophages preserve retinal ganglion cells and promote retinal progenitor cell renewal., The death of retinal ganglion cells (RGCs) is a hallmark of many retinal neuropathies. Neuroprotection, axonal regeneration, and cell renewal are vital for the integrity of the visual system after insult but are scarce in the adult mammalian retina. We hypothesized that monocyte-derived macrophages, known to promote healing in peripheral tissues, are required after an insult to the visual system, where their role has been largely overlooked. We found that after glutamate eye intoxication, monocyte-derived macrophages infiltrated the damaged retina of mice. Inhibition of this infiltration resulted in reduced survival of RGCs and diminished numbers of proliferating retinal progenitor cells (RPCs) in the ciliary body. Enhancement of the circulating monocyte pool led to increased RGC survival and RPC renewal. The infiltrating monocyte-derived macrophages skewed the milieu of the injured retina toward an antiinflammatory and neuroprotective one and down-regulated accumulation of other immune cells, thereby resolving local inflammation. The beneficial effect on RGC survival depended on expression of interleukin 10 and major histocompatibility complex class II molecules by monocyte-derived macrophages. Thus, we attribute to infiltrating monocyte-derived macrophages a novel role in neuroprotection and progenitor cell renewal in the injured retina, with far-reaching potential implications to retinal neuropathies and other neurodegenerative disorders.

The use of multi-modal optical diagnostic approach has a great potential in medicine and biology allowing significantly expand the capabilities of vascular diagnostics. Multi-modal approach provides synchronic in vivo images of blood and lymph vessels. In current study the Dynamic Light Scattering approach has been used for rendering blood vessels structure and monitoring blood micro-flows in the mouse ear in vivo with the higher spatial resolution, whereas conventional Fluorescence Intravital Microscopy provides simultaneous image of lymphatic vessels. The results clearly demonstrate that combined application of Dynamic Light Scattering and conventional Fluorescence Intravital Microscopy approaches provides synchronic in vivo images of blood and lymph vessels as well as blood micro-flows with a high contrast and specificity.

We combine diffusing wave spectroscopy and dynamic light scattering microscopy to simultaneous non-invasive imaging of skin blood microcirculation. We demonstrate for the first time to our knowledge that the local blood micro-flows and blood microcirculation can be observed and analyzed quantitatively at the the biological zero, when the arterial and vein flows are stopped completely. We show that the biological zero signal arises from the local blood micro-flows observed postmortem up to 100 minutes. The high sensitivity of diffusing correlation technique and the feasibility for non-invasive measurement of skin blood microcirculation is thus demonstrated, and the potential for methodology can now be explored.

Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd-DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material.

Background Antiangiogenic and anti-vascular therapies present intriguing alternatives to cancer therapy. However, despite promising preclinical results and significant delays in tumor progression, none have demonstrated long-term curative features to date. Here, we show that a single treatment session of Tookad-based vascular targeted photodynamic therapy (VTP) promotes permanent arrest of tumor blood supply by rapid occlusion of the tumor feeding arteries (FA) and draining veins (DV), leading to tumor necrosis and eradication within 24–48 h. Methodology/Principal Findings A mouse earlobe MADB106 tumor model was subjected to Tookad-VTP and monitored by three complementary, non-invasive online imaging techniques: Fluorescent intravital microscopy, Dynamic Light Scattering Imaging and photosensitized MRI. Tookad-VTP led to prompt tumor FA vasodilatation (a mean volume increase of 70%) with a transient increase (60%) in blood-flow rate. Rapid vasoconstriction, simultaneous blood clotting, vessel permeabilization and a sharp decline in the flow rates then followed, culminating in FA occlusion at 63.2 sec±1.5SEM. This blockage was deemed irreversible after 10 minutes of VTP treatment. A decrease in DV blood flow was demonstrated, with a slight lag from FA response, accompanied by frequent changes in flow direction before reaching a complete standstill. In contrast, neighboring, healthy tissue vessels of similar sizes remained intact and functional after Tookad-VTP. Conclusion/Significance Tookad-VTP selectively targets the tumor feeding and draining vessels. To the best of our knowledge, this is the first mono-therapeutic modality that primarily aims at the larger tumor vessels and leads to high cure rates, both in the preclinical and clinical arenas.

Introduction Necrosis at the tumor center is a common feature of aggressive breast cancers and has been associated with poor prognosis. It is commonly identified by means of invasive histopathology, which often correlates with morbidity and potential tumor cell dissemination, and limits the reconstruction of the whole necrotic domain. In this study we hypothesized that non covalent association to serum albumin (SA) and covalent binding to ligands for tumor-abundant cell receptors should synergistically drive selective accumulation and prolonged retention of imaging and therapeutic agents in breast tumor necrotic domains enabling in vivo identification, imaging and possibly treatment of such tumors. Methods Cyclo-Arg-Gly-Asp-D-Phe-Lys (c(RGDfK)) were conjugated to bacteriochlorophyll-derivatives (Bchl-Ds), previously developed as photodynamic agents, fluorescent probes and metal chelators in our lab. The c(RGDfK) component drives ligation to αVβ3 integrin receptors over-expressed by tumor cells and neo-vessels, and the Bchl-D component associates to SA in a non-covalent manner. STL-6014, a c(RGDfK)-Bchl-D representative, was i.v. injected to CD-1, nude female mice bearing necrotic and non-necrotic human MDA-MB-231-RFP breast cancer tumors. The fluorescence signals of the Bchl-Ds and RFP were monitored over days after treatment, by quantitative whole body imaging and excised tumor/tissue samples derived thereof. Complementary experiments included competitive inhibition of STL-6014 uptake by free c(RGDfK), comparative pharmacokinetics of nonconjugated c(RGDfK) Bchl-D (STL-7012) and of two human serum albumin (HSA) conjugates: HSA-STL-7012 and HSA-STL-6014. Results STL-6014 and STL-7012 formed complexes with HSA (HSA/STL-6014, HSA/STL-7012). STL-6014, HSA-STL-7012 and HSA-STL-6014, selectively accumulated at similar rates, in tumor viable regions over the first 8 h post administration. They then migrated into the necrotic tumor domain and presented tumor half lifetimes (T1/2) in the range of days where T1/2 for HSA-STL-6014 > STL-6014 > HSA-STL-7012. No accumulation of STL-7012 was observed. Pre-injection of c(RGDfK) excess, prevented the uptake of STL-6014 in the small, but not in the large tumors. Conclusions Non-covalent association to SA and covalent binding to c(RGDfK), synergistically enable the accumulation and prolonged retention of Bchl-Ds in the necrotic regions of tumors. These findings provide novel guidelines and strategy for imaging and treatment of necrotic tumors.

Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)–based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells. [Cancer Res 2009;69(13):5610–7]

Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell-deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-beta1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.

Angiogenesis is a multistep cellular process in which new vessels emerge from preexisting endothelial vasculature in the presence of various growth factors and extracellular matrix (ECM) proteins (...

This study hypothesized that success rate assessment of vascular targeted photodynamic therapy (VTP) of solid tumors 24 h posttreatment may allow prompt administration of a second treatment in case of failure, increasing the overall success rate. Here, we show that treatment of luciferase transfected CT26-luc mouse colon carcinoma tumors in BALB ⁄ c mice by VTP with WST11 (a Pd-bacteriochlorophyll-based photosensitizer) allows fast assessment of treatment success 24 h post-treatment, using bioluminescence imaging (BLI). WST11-VTP was found to abolish luciferin bioluminescence in the treated tumors resulting in two types of responses. One, comprising 75% of the mice, signified successful outcome, presenting neither BLI signal nor tumor regrowth (24 h–90 days post-VTP). The second (the remaining 25% of the mice) signified treatment failure, presenting various levels of BLI signal with subsequent tumor regrowth (24 h–90 days). Consequently, the mice that failed the first treatment were treated again. We show that treatment success rate in both VTP sessions was identical and that the cumulative success rate of the treatment increased from 75% to over 90%. These results therefore, present a fast method of assessing VTP outcome and support the feasibility of successive multiple treatments with these sensitizers in the clinical arena. The presented methodology can also be helpful in future preclinical studies, and expedite the development of VTP drugs.

Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.

Physiological blood coagulation is an essential biological process. Current tests for plasma coagulation (clotting) need to be performed ex vivo and require fresh blood sampling for every test. A recently published work describes a new, noninvasive, in vivo approach to assess blood coagulation status during mechanical occlusion1. For this purpose, we have tested this approach and applied a controlled laser beam to blood micro-vessels of the mouse ear during mechanical occlusion. Standard setup for intravital transillumination videomicroscopy and laser based imaging techniques were used for monitoring the blood clotting process. Temporal mechanical occlusion of blood vessels in the observed area was applied to ensure blood flow cessation. Subsequently, laser irradiation was used to induce vascular micro-injury. Changes in the vessel wall, as well as in the pattern of blood flow, predispose the area to vascular thrombosis, according to the paradigm of Virchow's triad. In our experiments, two elements of Virchow's triad were used to induce the process of clotting in vivo, and to assess it optically. We identified several parameters that can serve as markers of the blood clotting process in vivo. These include changes in light absorption in the area of illumination, as well as changes in the pattern of the red blood cells' micro-movement in the vessels where blood flow is completely arrested. Thus, our results indicate that blood coagulation status can be characterized by non-invasive, in vivo methodologies.

The use of small animals in intravital optical microscopy is a well-established experimental model to study blood microcirculation in vivo . Recent advances in cell biology and optical techniques (e.g., lasers, CCD cameras, software, etc.) provide the basis for significant improvements with in vivo imaging. This review summarizes the latest achievements in this specific area focusing on the development of modern optical and biological platforms. This includes in vivo real time monitoring of individual cells in the context of blood flow, super-sensitive fluorescence imaging, high-speed cell imaging and light scattering techniques. The capability of these platforms has been demonstrated in live animal models (e.g., mouse and rat ear, rat mesentery, and others) for real-time monitoring of individual blood cell properties (e.g., size and shape), cell trafficking, cell-cell interactions (e.g., aggregation in flow or adhesion to vessel walls), and blood flow viscosity. Future applications are discussed including in vivo early diagnosis of disease and monitoring cellular responses to environmental and therapeutic interventions.

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.

Physiological blood coagulation/clotting is an essential biological process that is initiated by vessel injury and includes a cascade of enzymatic reactions finalized by fibrin polymerization and clot formation. We utilize dynamic light scattering (DLS) imaging to monitor in vivo red cell mobility as an indicator of blood coagulation. In the course of the experiments, blood flow is arrested using mechanical occlusion, and then laser injury is applied. We demonstrate that the combination of laser injury with DLS imaging on occluded blood vessels (i.e., under static conditions) is suitable to detect even subtle changes of plasma viscosity in the circulatory system, which reflects the process of clot development. This approach is noninvasive and has a relatively simple and easy-to-use technical design. Thus, the proposed methodology provides a promising tool for investigating blood clotting within the vasculature.

Vascular endothelial growth factor C (VEGF-C) plays a critical role in tumor lymphangiogenesis and lymph node metastasis. We report here that VEGF-C expression is regulated by microenvironmental stress including hyperthermia and oxidative stress. Furthermore, we show that this stress response is mediated by transcriptional activation mediated by lens epithelium-derived growth factor (LEDGF/p75). Ectopic expression of LEDGF/p75 in C6 rat glioma and in H1299 human non-small cell lung carcinoma induced VEGF-C expression in vitro, whereas in subcutaneous mouse tumor xenografts, LEDGF/p75 stimulated VEGF-C expression and augmented angiogenesis and lymphangiogenesis. Conversely, overexpression of a LEDGF/p75 native antisense or LEDGF/p75-targeted short interfering RNA downmodulated VEGF-C expression. LEDGF seemed to conferred this activity on binding to a conserved stress response element (STRE) located in the VEGF-C gene because mutating the STRE was sufficient for the suppression of basal and stress-induced activations of the VEGF-C promoter. Thus, the study reported here identified a role for LEDGF/p75 in stress-regulated transcriptional control of VEGF-C expression. These results provide a possible link for LEDGF/p75 in tumor lymphangiogenesis and cancer metastasis. Hence, our data suggest the LEDGF-VEGF-C axis as a putative biomarker for the detection of stress-induced lymphangiogenesis and LEDGF as a potential target for antimetastatic therapy.