Spatial determinates of effector and memory CD8+ T cell fates.

The lymph node plays a critical role in mounting an adaptive immune response to infection, clearance of foreign pathogens, and cancer immunosurveillance. Within this complex structure, intranodal migration is vital for CD8+ T cell activation and differentiation. Combining tissue clearing and volumetric light sheet fluorescent microscopy of intact lymph nodes has allowed us to explore the spatial regulation of T cell fates. This has determined that short-lived effector (TSLEC ) are imprinted in peripheral lymph node interfollicular regions, due to CXCR3 migration. In contrast, stem-like memory cell (TSCM ) differentiation is determined in the T cell paracortex. Here, we detail the inflammatory and chemokine regulators of spatially restricted T cell differentiation, with a focus on how to promote TSCM . We propose a default pathway for TSCM differentiation due to CCR7-directed segregation of precursors away from the inflammatory effector niche. Although volumetric imaging has revealed the consequences of intranodal migration, we still lack knowledge of how this is orchestrated within a complex chemokine environment. Toward this goal, we highlight the potential of combining microfluidic chambers with pre-determined complexity and subcellular resolution microscopy.

Skinny deeping: Uncovering immune cell behavior and function through imaging techniques.

As the largest organ of the body, the skin is a key barrier tissue with specialized structures where ongoing immune surveillance is critical for protecting the body from external insults. The innate immune system acts as first-responders in a coordinated manner to react to injury or infections, and recent developments in intravital imaging techniques have made it possible to delineate dynamic immune cell responses in a spatiotemporal manner. We review here key studies involved in understanding neutrophil, dendritic cell and macrophage behavior in skin and further discuss how this knowledge collectively highlights the importance of interactions and cellular functions in a systems biology manner. Furthermore, we will review emerging imaging technologies such as high-content proteomic screening, spatial transcriptomics and three-dimensional volumetric imaging and how these techniques can be integrated to provide a systems overview of the immune system that will further our current knowledge and lead to potential exciting discoveries in the upcoming decades.

Imaging leukocyte migration through afferent lymphatics.

Afferent lymphatics mediate the transport of antigen and leukocytes, especially of dendritic cells (DCs) and T cells, from peripheral tissues to draining lymph nodes (dLNs). As such they play important roles in the induction and regulation of adaptive immunity. Over the past 15 years, great advances in our understanding of leukocyte trafficking through afferent lymphatics have been made through time-lapse imaging studies performed in tissue explants and in vivo, allowing to visualize this process with cellular resolution. Intravital imaging has revealed that intralymphatic leukocytes continue to actively migrate once they have entered into lymphatic capillaries, as a consequence of the low flow conditions present in this compartment. In fact, leukocytes spend considerable time migrating, patrolling and interacting with the lymphatic endothelium or with other intralymphatic leukocytes within lymphatic capillaries. Cells typically only start to detach once they arrive in downstream-located collecting vessels, where vessel contractions contribute to enhanced lymph flow. In this review, we will introduce the biology of afferent lymphatic vessels and report on the presumed significance of DC and T cell migration via this route. We will specifically highlight how time-lapse imaging has contributed to the current model of lymphatic trafficking and the emerging notion that - besides transport - lymphatic capillaries exert additional roles in immune modulation.

Imaging white matter microstructure with gradient-echo phase imaging: Is ex vivo imaging with formalin-fixed tissue a good approximation of the in vivo brain?

PURPOSE: Ex vivo imaging is a commonly used approach to investigate the biophysical mechanism of orientation-dependent signal phase evolution in white matter. Yet, how phase measurements are influenced by the structural alteration in the tissue after formalin fixation is not fully understood. Here, we study the effects on magnetic susceptibility, microstructural compartmentalization, and chemical exchange measurement with a postmortem formalin-fixed whole-brain human tissue. METHODS: A formalin-fixed, postmortem human brain specimen was scanned with multiple orientations to the main magnetic field direction for robust bulk magnetic susceptibility measurement with conventional quantitative susceptibility imaging models. White matter samples were subsequently excised from the whole-brain specimen and scanned in multiple rotations on an MRI scanner to measure the anisotropic magnetic susceptibility and microstructure-related contributions in the signal phase and to validate the findings of the whole-brain data. RESULTS: The bulk isotropic magnetic susceptibility of ex vivo whole-brain imaging is comparable to in vivo imaging, with noticeable enhanced nonsusceptibility contributions. The excised specimen experiment reveals that anisotropic magnetic susceptibility and compartmentalization phase effect were considerably reduced in the formalin-fixed white matter specimens. CONCLUSIONS: Formalin-fixed postmortem white matter exhibits comparable isotropic magnetic susceptibility to previous in vivo imaging findings. However, the measured phase and magnitude data of the fixed white matter tissue shows a significantly weaker orientation dependency and compartmentalization effect. Alternatives to formalin fixation are needed to better reproduce the in vivo microstructural effects in postmortem samples.

Imaging of mast cells.

Mast cells are a part of the innate immune system implicated in allergic reactions and the regulation of host-pathogen interactions. The distribution, morphology and biochemical composition of mast cells has been studied in detail in vitro and on tissue sections both at the light microscopic and ultrastructural level. More recently, the development of fluorescent reporter strains and intravital imaging modalities has enabled first glimpses of the real-time behavior of mast cells in situ. In this review, we describe commonly used imaging approaches to study mast cells in cell culture as well as within normal and diseased tissues. We further describe the interrogation of mast cell function via imaging by providing a detailed description of mast cell-nerve plexus interactions in the intestinal tract. Together, visualizing mast cells has expanded our view of these cells in health and disease.

Tumor Accumulation and Off-Target Biodistribution of an Indocyanine-Green Fluorescent Nanotracer: An Ex Vivo Study on an Orthotopic Murine Model of Breast Cancer.

Indocyanine green (ICG) is a near infrared fluorescent tracer used in image-guided surgery to assist surgeons during resection. Despite appearing as a very promising tool for surgical oncology, its employment in this area is limited to lymph node mapping or to laparoscopic surgery, as it lacks tumor targeting specificity. Recently, a nanoformulation of this dye has been proposed with the aim toward tumor targeting specificity in order to expand its employment in surgical oncology. This nanosystem is constituted by 24 monomers of H-Ferritin (HFn), which self-assemble into a spherical cage structure enclosing the indocyanine green fluorescent tracer. These HFn nanocages were demonstrated to display tumor homing due to the specific interaction between the HFn nanocage and transferrin receptor 1, which is overexpressed in most tumor tissues. Here, we provide an ex vivo detailed comparison between the biodistribution of this nanotracer and free ICG, combining the results obtained with the Karl Storz endoscope that is currently used in clinical practice and the quantification of the ICG signal derived from the fluorescence imaging system IVIS Lumina II. These insights demonstrate the suitability of this novel HFn-based nanosystem in fluorescence-guided oncological surgery.

Imaging of the ex vivo transglutaminase activity in liver macrophages of sepsis mice.

Sepsis is the leading cause of death in hospitalized patients and is characterized by a dysregulated inflammatory response to infection and multiple organ failure, including the liver. Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, and integrin-binding activities and has opposing roles in the regulation of cell growth, differentiation, and apoptosis. TG2 plays both pathogenic and protective roles in liver diseases, revealing the need to examine the activities of TG2. Here, we introduced an ex vivo imaging approach to examine the in vivo transamidase activity of TG2 based on the combination of intraperitoneal injection of 5-biotinamidopentylamine (5BAPA), a biotinylated substrate for TG2, and fluorescent streptavidin staining in frozen liver sections. Increased 5BAPA signals was observed in the livers of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced sepsis mice. Pharmacological inhibition of TG2 activity ameliorated LPS-induced liver injury. 5BAPA signals were observed in TG2-expressing and F4/80-positive midzonal macrophages, providing direct evidence that activated macrophages are the major cellular source of active TG2 in the livers of sepsis mice. Further studies focusing on the activation of 5BAPA-stained midzonal macrophages may improve understanding of the molecular pathophysiology and the development of therapeutic strategies for sepsis.

The choice of embedding media affects image quality, tissue R2* , and susceptibility behaviors in post-mortem brain MR microscopy at 7.0T.

PURPOSE: The quality and precision of post-mortem MRI microscopy may vary depending on the embedding medium used. To investigate this, our study evaluated the impact of 5 widely used media on: (1) image quality, (2) contrast of high spatial resolution gradient-echo (T1 and T2* -weighted) MR images, (3) effective transverse relaxation rate (R2* ), and (4) quantitative susceptibility measurements (QSM) of post-mortem brain specimens. METHODS: Five formaldehyde-fixed brain slices were scanned using 7.0T MRI in: (1) formaldehyde solution (formalin), (2) phosphate-buffered saline (PBS), (3) deuterium oxide (D2 O), (4) perfluoropolyether (Galden), and (5) agarose gel. SNR and contrast-to-noise ratii (SNR/CNR) were calculated for cortex/white matter (WM) and basal ganglia/WM regions. In addition, median R2* and QSM values were extracted from caudate nucleus, putamen, globus pallidus, WM, and cortical regions. RESULTS: PBS, Galden, and agarose returned higher SNR/CNR compared to formalin and D2 O. Formalin fixation, and its use as embedding medium for scanning, increased tissue R2* . Imaging with agarose, D2 O, and Galden returned lower R2* values than PBS (and formalin). No major QSM offsets were observed, although spatial variance was increased (with respect to R2* behaviors) for formalin and agarose. CONCLUSIONS: Embedding media affect gradient-echo image quality, R2* , and QSM in differing ways. In this study, PBS embedding was identified as the most stable experimental setup, although by a small margin. Agarose and Galden were preferred to formalin or D2 O embedding. Formalin significantly increased R2* causing noisier data and increased QSM variance.

How Nanoparticle Physicochemical Parameters Affect Drug Delivery to Cells in the Retina via Systemic Interactions.

Minor changes in the composition of poloxamer 188-modified, DEAE-dextran-stabilized (PDD) polybutylcyanoacrylate (PBCA) nanoparticles (NPs), by altering the physicochemical parameters (such as size or surface charge), can substantially influence their delivery kinetics across the blood-retina barrier (BRB) in vivo. We now investigated the physicochemical mechanisms underlying these different behaviors of NP variations at biological barriers and their influence on the cellular and body distribution. Retinal whole mounts from rats injected in vivo with fluorescent PBCA NPs were processed for retina imaging ex vivo to obtain a detailed distribution of NPs with cellular resolution in retinal tissue. In line with previous in vivo imaging results, NPs with a larger size and medium surface charge accumulated more readily in brain tissue, and they could be more easily detected in retinal ganglion cells (RGCs), demonstrating the potential of these NPs for drug delivery into neurons. The biodistribution of the NPs revealed a higher accumulation of small-sized NPs in peripheral organs, which may reduce the passage of these particles into brain tissue via a "steal effect" mechanism. Thus, systemic interactions significantly determine the potential of NPs to deliver markers or drugs to the central nervous system (CNS). In this way, minor changes of NPs' physicochemical parameters can significantly impact their rate of brain/body biodistribution.

Intratumoral delivery and therapeutic efficacy of nanoparticle-encapsulated anti-tumor siRNA following intrapulmonary administration for potential treatment of lung cancer.

This study evaluated the delivery efficiency and antitumor effects of the intrapulmonary administration of antitumor small interfering ribonucleic acid (siRNA)-containing nanoparticles to mice with metastatic lung tumor. Fluorescence-labeled, siRNA-containing nanoparticles were administered using Liquid MicroSprayer® to mice with metastatic lung tumors induced by the murine melanoma cell line B16F10. Fluorescent signals in the whole lung and in the tumor region following the intrapulmonary administration of siRNA-containing nanoparticles were stronger than those following intravenous administration. The intrapulmonary administration of nanoparticles containing a mixture of siRNA against MDM2, c-Myc, and vascular endothelial growth factor (VEGF) significantly improved survival and prolonged the survival of mice with metastatic lung tumor. In addition, after the intrapulmonary or intravenous administration of the mixture, the activity levels of interleukin-6 and -12, markers of systemic toxicity, were similar to those of nontreatment. These results indicate that the antitumor siRNA-containing nanoparticles were delivered efficiently and specifically to tumor cells, effectively silencing the oncogenes in the lung metastasis without any significant systemic toxicity.

A Novel Fluorescent Quantum Dot Probe for the Rapid Diagnostic High Contrast Imaging of Tumor in Mice.

A simple probe - antibody conjugated silica over coated cadmium selenide quantum dots (QD-Ab probe) for efficient and rapid diagnostic in vivo imaging of tumors is developed. Compared to unconjugated quantum dots (QD), these probes underwent efficient cellular internalization and tumor targeting behavior, retaining bright emission under in vivo cancer models. Silica over coated cadmium selenide quantum dots were conjugated with Epidermal growth factor receptor (EGFR) monoclonal antibody to detect the over expression of EGFR in cancer models. The in vitro cellular internalization efficiency of QD and QD-Ab probe in cultured stem cells (RADMSCs) and cancer cells (HeLa) were assessed by ICP-OES and cLSM. Results demonstrated a greater internalization efficiency of CdSe-Silica QD-Ab probe than CdSe-Silica QDs. For in vivo imaging solid tumor bearing mice was subjected to tail vein injection of QD and QD-Ab probe. After the specific time interval of injection, mice were anesthetized and subjected into Xenogen IVIS®200 imaging system, followed by ex vivo imaging. Subsequently, ultrathin sections of tumor were imaged by using cLSM. Both in vivo and ex vivo imaging results confirmed the tumor-targeted imaging efficiency of QD-Ab probes compared to unconjugated QDs.

Q-space truncation and sampling in diffusion spectrum imaging.

PURPOSE: To characterize the q-space truncation and sampling on the spin-displacement probability density function (PDF) in diffusion spectrum imaging (DSI). METHODS: DSI data were acquired using the MGH-USC connectome scanner (Gmax = 300 mT/m) with bmax = 30,000 s/mm2 , 17 × 17 × 17, 15 × 15 × 15 and 11 × 11 × 11 grids in ex vivo human brains and bmax = 10,000 s/mm2 , 11 × 11 × 11 grid in vivo. An additional in vivo scan using bmax =7,000 s/mm2 , 11 × 11 × 11 grid was performed with a derated gradient strength of 40 mT/m. PDFs and orientation distribution functions (ODFs) were reconstructed with different q-space filtering and PDF integration lengths, and from down-sampled data by factors of two and three. RESULTS: Both ex vivo and in vivo data showed Gibbs ringing in PDFs, which becomes the main source of artifact in the subsequently reconstructed ODFs. For down-sampled data, PDFs interfere with the first replicas or their ringing, leading to obscured orientations in ODFs. CONCLUSION: The minimum required q-space sampling density corresponds to a field-of-view approximately equal to twice the mean displacement distance (MDD) of the tissue. The 11 × 11 × 11 grid is suitable for both ex vivo and in vivo DSI experiments. To minimize the effects of Gibbs ringing, ODFs should be reconstructed from unfiltered q-space data with the integration length over the PDF constrained to around the MDD. Magn Reson Med 76:1750-1763, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Robust methods to create ex vivo minimum deformation atlases for brain mapping.

Highly detailed ex vivo 3D atlases of average structure are of critical importance to neuroscience and its current push to understanding the global microstructure of the brain. Multiple single slice histology sections can no longer provide sufficient detail of inter-slice microstructure and lack out of plane resolution. Two ex vivo methods have emerged that can create such detailed models. High-field micro MRI with the addition of contrast media has allowed intact whole brain microstructure imaging with an isotropic resolution of 15 µm in mouse. Blockface imaging has similarly evolved to a point where it is now possible to image an entire brain in a rigorous fashion with an out of plane resolution of 10 µm. Despite the destruction of the tissue as part of this process it allows a reconstructed model that is free from cutting artifacts. Both of these methods have been utilised to create minimum deformation atlases that are representative of the respective populations. The MDA atlases allow us unprecedented insight into the commonality and differences in microstructure in cortical structures in specific taxa. In this paper we provide an overview of how to create such MDA models from ex vivo data.

Biodistribution and in vivo activities of tumor-associated macrophage-targeting nanoparticles incorporated with doxorubicin.

Tumor-associated macrophages (TAMs) are increasingly considered a viable target for tumor imaging and therapy. Previously, we reported that innovative surface-functionalization of nanoparticles may help target them to TAMs. In this report, using poly(lactic-co-glycolic) acid (PLGA) nanoparticles incorporated with doxorubicin (DOX) (DOX-NPs), we studied the effect of surface-modification of the nanoparticles with mannose and/or acid-sensitive sheddable polyethylene glycol (PEG) on the biodistribution of DOX and the uptake of DOX by TAMs in tumor-bearing mice. We demonstrated that surface-modification of the DOX-NPs with both mannose and acid-sensitive sheddable PEG significantly increased the accumulation of DOX in tumors, enhanced the uptake of the DOX by TAMs, but decreased the distribution of DOX in mononuclear phagocyte system (MPS), such as liver. We also confirmed that the acid-sensitive sheddable PEGylated, mannose-modified DOX-nanoparticles (DOX-AS-M-NPs) targeted TAMs because depletion of TAMs in tumor-bearing mice significantly decreased the accumulation of DOX in tumor tissues. Furthermore, in a B16-F10 tumor-bearing mouse model, we showed that the DOX-AS-M-NPs were significantly more effective than free DOX in controlling tumor growth but had only minimum effect on the macrophage population in mouse liver and spleen. The AS-M-NPs are promising in targeting cytotoxic or macrophage-modulating agents into tumors to improve tumor therapy.

Brain abscess - A rare confounding factor for diagnosis of post-traumatic epilepsy after lateral fluid-percussion injury.

OBJECTIVE: To assess the prevalence of brain abscesses as a confounding factor for the diagnosis of post-traumatic epilepsy (PTE) in a rat model of lateral fluid-percussion-induced (FPI) traumatic brain injury (TBI). METHODS: This retrospective study included 583 rats from 3 study cohorts collected over 2009-2022 in a single laboratory. The rats had undergone sham-operation or TBI using lateral FPI. Rats were implanted with epidural and/or intracerebral electrodes for electroencephalogram recordings. Brains were processed for histology to screen for abscess(es). In abscess cases, (a) unfolded cortical maps were constructed to assess the cortical location and area of the abscess, (b) the abscess tissue was Gram stained to determine the presence of gram-positive and gram-negative bacteria, and (c) immunostaining was performed to detect infiltrating neutrophils, T-lymphocytes, and glial cells as tissue biomarkers of inflammation. In vivo and/or ex vivo magnetic resonance images available from a subcohort of animals were reviewed to evaluate the presence of abscesses. Plasma samples available from a subcohort of rats were used for enzyme-linked immunosorbent assays to determine the levels of lipopolysaccharide (LPS) as a circulating biomarker for gram-negative bacteria. RESULTS: Brain abscesses were detected in 2.6% (15/583) of the rats (6 sham, 9 TBI). In histology, brain abscesses were characterized as vascularized encapsulated lesions filled with neutrophils and surrounded by microglia/macrophages and astrocytes. The abscesses were mainly located under the screw electrodes, support screws, or craniectomy. Epilepsy was diagnosed in 60% (9/15) of rats with an abscess (4 sham, 5 TBI). Of these, 67% (6/9) had seizure clusters. The average seizure frequency in abscess cases was 0.436 ± 0.281 seizures/d. Plasma LPS levels were comparable between rats with and without abscesses (p > 0.05). SIGNIFICANCE: Although rare, a brain abscess is a potential confounding factor for epilepsy diagnosis in animal models of structural epilepsies following brain surgery and electrode implantation, particularly if seizures occur in sham-operated experimental controls and/or in clusters.

A portable photoacoustic microscopy and ultrasound system for rectal cancer imaging.

Photoacoustic microscopy offers functional information regarding tissue vasculature while ultrasound characterizes tissue structure. Combining these two modalities provides novel clinical applications including response assessment among rectal cancer patients undergoing therapy. We have previously demonstrated the capabilities of a co-registered photoacoustic and ultrasound device in vivo, but multiple challenges limited broad adoption. In this paper, we report significant improvements in an acoustic resolution photoacoustic microscopy and ultrasound (ARPAM/US) system characterized by simulation and phantom study, focusing on resolution, optical coupling, and signal characteristics. In turn, higher in-probe optical coupling efficiency, higher signal-to-noise ratio, higher data throughput, and better stability with minimal maintenance requirements were all accomplished. We applied the system to 19 ex vivo resected colorectal cancer samples and found significantly different signals between normal, cancer, and post-treatment tumor tissues. Finally, we report initial results of the first in vivo imaging study.

Visualizing the Cell-Matrix Interactions and Cytoskeleton of Disseminated Tumor Cells.

Tumor cells often leave the primary tumor mass and get settled in a foreign tissue years before the development of overt metastases, exhibiting the highly inefficient nature of metastatic colony formation. In fact, the tumor cells that disseminate into distant organs and subsequently invade the parenchyma of these organs rarely proceed to found actively growing metastatic colonies. Instead, the majority of these tumor cells undergo prolonged proliferative arrest unless they are swiftly eliminated by the immune system. Together, these observations indicate that the proliferative capacity of the disseminated tumor cells (DTCs) serves as a key determinant of the efficiency of metastasis, highlighting the need to better understand the mechanism governing the proliferation of these cells. Recent studies are unveiling the importance of the interactions between DTCs and the microenvironment of the host tissue in regulating the proliferation of DTCs. However, the details of such interactions remain to be fully delineated. Here I describe the methods for visualizing and analyzing the interactions between DTCs and the extracellular matrix (ECM) components of the host tissue as well as the cytoskeleton of the DTCs that support these interactions. The methods described here will facilitate the study of how DTCs interact with the ECM of their host tissue, which will be crucial for elucidating the mechanism that underlies the regulation of DTC proliferation by the DTC-ECM interactions.

APOE4 genotype and aging impair injury-induced microglial behavior in brain slices, including toward Aß, through P2RY12.

Microglia are highly dynamic cells that play a critical role in tissue homeostasis through the surveillance of brain parenchyma and response to cues associated with damage. Aging and APOE4 genotype are the strongest risk factors for Alzheimer's disease (AD), but how they affect microglial dynamics remains unclear. Using ex vivo confocal microscopy, we analyzed microglial dynamic behaviors in the entorhinal cortex (EC) and hippocampus CA1 of 6-, 12-, and 21-month-old mice APOE3 or APOE4 knock-in mice expressing GFP under the CX3CR1 promoter. To study microglia surveillance, we imaged microglia baseline motility for 20 min and measured the extension and retraction of processes. We found that APOE4 microglia exhibited significantly less brain surveillance (27%) compared to APOE3 microglia in 6-month-old mice; aging exacerbated this deficit. To measure microglia response to damage, we imaged process motility in response to ATP, an injury-associated signal, for 30 min. We found APOE4 microglia extended their processes significantly slower (0.9 µm/min, p < 0.005) than APOE3 microglia (1.1 µm/min) in 6-month-old animals. APOE-associated alterations in microglia motility were observed in 12- and 21-month-old animals, and this effect was exacerbated with aging in APOE4 microglia. We measured protein and mRNA levels of P2RY12, a core microglial receptor required for process movement in response to damage. We found that APOE4 microglia express significantly less P2RY12 receptors compared to APOE3 microglia despite no changes in P2RY12 transcripts. To examine if the effect of APOE4 on the microglial response to ATP also applied to amyloid ß (Aß), we infused locally Hi-Lyte Fluor 555-labeled Aß in acute brain slices of 6-month-old mice and imaged microglia movement for 2 h. APOE4 microglia showed a significantly slower (p < 0.0001) process movement toward the Aß, and less Aß coverage at early time points after Aß injection. To test whether P2RY12 is involved in process movement in response to Aß, we treated acute brain slices with a P2RY12 antagonist before Aß injection; microglial processes no longer migrated towards Aß. These results provide mechanistic insights into the impact of APOE4 genotype and aging in dynamic microglial behaviors prior to gross Aß pathology and could help explain how APOE4 brains are more susceptible to AD pathogenesis.

Ex vivo fluorescence imaging for the identification of rhodamine-labeled bovine serum albumin and chitosan-coated gold and silver nanoparticles.

Therapeutic potential and toxic effects of in vivo administered gold nanoparticles (GNPs) and silver nanoparticles (SNP) depend on distribution in tissues. Rhodamine (Rho) labeled bovine serum albumin (BSA) and chitosan (Chi) were prepared by covalent conjugation and were characterized by fluorescence spectral analysis. GNP and SNP were coated with the labeled conjugates of BSA and chitosan by adsorption. The soluble Rho-BSA or Rho-Chi conjugates, uncoated, and conjugate-coated GNP, and SNP were orally administered into 8-week-old rats. After 24 h, rats were euthanized and the liver, kidney, spleen, and thymus were dissected. The tissues were examined ex vivo using a small animal in vivo imaging system. The liver, kidney, and thymus displayed higher fluorescence due to increased accumulation of Rho-BSA or Rho-Chi conjugate-coated nanoparticles (NPs) in the tissues as compared to the spleen where lower fluorescence was noticed. Tissues obtained from rats that were administered Rho-BSA or Rho-Chi conjugate-coated GNP and SNP showed tenfold higher fluorescence intensity as compared to tissues from rats that were given soluble conjugates or NP alone. The results strongly suggest significant tissue distribution of NP following oral administration.

Use of Freshly Amputated Human Limbs for Pre-Clinical Evaluation of Molecular-Targeted Fluorescent Probes.

We have co-developed a first-in-kind model of fluorophore testing in freshly amputated human limbs. Ex vivo human tissue provides a unique opportunity for the testing of pre-clinical fluorescent agents, collection of imaging data, and histopathologic examination in human tissue prior to performing in vivo experiments. Existing pre-clinical fluorescent agent studies rely primarily on animal models, which do not directly predict fluorophore performance in humans and can result in wasted resources and time if an agent proves ineffective in early human trials. Because fluorophores have no desired therapeutic effect, their clinical utility is based solely on their safety and ability to highlight tissues of interest. Advancing to human trials even via the FDA's phase 0/microdose pathway still requires substantial resources, single-species pharmacokinetic testing, and toxicity testing. In a recently concluded study using amputated human lower limbs, we were able to test successfully a nerve-specific fluorophore in pre-clinical development. This study used systemic administration via vascular cannulization and a cardiac perfusion pump. We envision that this model may assist with early lead agent testing selection for fluorophores with various targets and mechanisms.