KEYWORDS: Luminescence, In vivo imaging, Collagen, Inflammation, Imaging systems, Visualization, Animal model studies, Near infrared, Pathology, Cartilage
An in vivo near-infrared fluorescence (NIRF) imaging technique is described for therapy monitoring of ankle joints affected by collagen-induced arthritis, a model of human rheumatoid arthritis. Arthritis was induced in rats by intradermal injections of collagen and Freund’s incomplete adjuvant. For in vivo imaging, the nonspecific NIR dye tetrasulfocyanine (TSC) was used. Prior to and after treatment with a nonsteroidal anti-inflammatory drug, meloxicam, or analgesic drug, tramadol hydrochloride (which served as no-therapy control), normalized fluorescence intensities of each ankle joint were measured. Additionally, each ankle joint was characterized by clinical arthritis scoring and histopathology. Over a 3-week treatment period, a significant difference in disease progression between animals treated with meloxicam and tramadol hydrochloride was detected. A statistically significant improvement in ankle joint pathology from high- or moderate-grade to moderate- or low-grade upon meloxicam therapy, as determined by clinical evaluation, translated into a significant decrease in fluorescence intensity. In contrast, all arthritic joints of the no-therapy control group deteriorated to high-grade arthritis with high-fluorescence intensities in NIRF imaging.
KEYWORDS: Magnetic resonance imaging, Luminescence, Collagen, Signal to noise ratio, Inflammation, Animal model studies, Statistical analysis, Plasma, Absorption, Imaging systems
Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (I NF ) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. I NF of moderate and high-grade arthritic joints were significantly higher (p<0.005 ) than the values of control and low-grade arthritic joints between 5 and 30 min after TSC-injection. This result correlated well with post-contrast MRI signal intensities at about 5 min after gadodiamide administration. Furthermore, I NF and signal increase on contrast-enhanced MRI showed high correlation with clinical and histopathological scores. Sensitivities and specificities for detection of moderate and high-grade arthritic joints were slightly lower for NIRF imaging (89%/81% ) than for MRI (100%/91% ). NIRF imaging using TSC, which is characterized by slower plasma clearance compared to indocyanine green (ICG), has the potential to improve monitoring of inflamed joints.
Thomas Dziekan, Carmen Weissbach, Jan Voigt, Bernd Ebert, Rainer Macdonald, Malte Bahner, Marianne Mahler, Michael Schirner, Michael Berliner, Birgitt Berliner, Jens Osel, Ilka Osel
Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.
We report on the development of a scanning non-contact brain imager, based on a novel technique in time-resolved nearinfrared
spectroscopy, i.e. the null source-detector distance approach. Our concept is designed to image an area of about
10 cm2 with small adjustable scanning steps, i.e. a high density of mapping points can be realized. The feasibility of the
proposed method was tested with a single-point confocal optical setup without beam scanning so far. A set of test
measurements was performed on a liquid phantom with a small black polyvinyl chloride (PVC) cylinder as a target,
which was translated in X direction to emulate the optical scanning and estimate lateral spatial resolution, and in Z
direction to estimate the depth sensitivity of the instrument. The problem of dominance of early photons at null sourcedetector
separation was solved by applying a fast time-gated detector to detect late only photons. Two fast-gated
detectors, a newly developed state-of-art time-gated single-photon avalanche photodiode (tgSPAD) and commercially
available fast-gated intensified CCD (iCCD) camera, were compared against each other. It was shown that, due to its
better dynamic range, the tgSPAD is capable to detect later photons than the iCCD camera, and hence, a scanning system
equipped with the time-gated SPAD has better depth sensitivity. Thus the time-gated SPAD is the detector of choice for
further development of the non-contact confocal brain scanner.
We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1′-bis-(4-sulfobutyl) indotricarbocyanine-5,5′-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.
Bernd Ebert, Thomas Dziekan, Carmen Weissbach, Marianne Mahler, Michael Schirner, Birgitt Berliner, Daniel Bauer, Jan Voigt, Michael Berliner, Malte L. Bahner, Rainer Macdonald
The blood pool agent indo-cyanine green (ICG) has been investigated in a prospective clinical study for detection of
rheumatoid arthritis using fluorescence imaging. Temporal behavior as well as spatial distribution of fluorescence
intensity are suited to differentiate healthy and inflamed finger joints after i.v. injection of an ICG bolus.
KEYWORDS: Luminescence, Scanners, Imaging systems, Magnetic resonance imaging, In vivo imaging, Scattering, Near infrared, Spatial resolution, Animal model studies, Head
We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR)
spectral range following intravenous administration of contrast agents. The new imaging system has been characterized
with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments
were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint
inflammation have been compared with findings from contrast enhanced MR imaging and histology.
Kai Licha, N. Debus, S. Emig-Vollmer, B. Hofmann, M. Hasbach, D. Stibenz, S. Sydow, Michael Schirner, Bernd Ebert, Diethard Petzelt, C. Bührer, W. Semmler, R. Tauber
We develop a highly specific antibody-dye conjugate for optical imaging of peripheral lymph nodes. The contrast agent consists of the monoclonal antibody recognizing endothelial ligands for the lymphocyte homing receptor L-selectin, MECA-79, and a near-infrared (near-IR) fluorescent indotricarbocyanine dye. The targeting and biodistribution behavior of MECA-79 is studied after radio-iodination and intravenous injection into mice demonstrating specific uptake in lymph nodes and accumulation in high endothelial venules (HEV). After conjugation of MECA-79 with indotricarbocyanine dye, the fluorescence imaging properties of the MECA-79 dye conjugate are examined by intravenous injection in nude mice and laser-induced fluorescence whole-body imaging in vivo. The MECA-79 antibody-dye conjugate accumulates in peripheral lymph nodes, whereas an isotype antibody-dye conjugate does not. Specific lymph node near-IR fluorescent signals become detectable within minutes after injection, and stable imaging persists for more than 24 h. The results demonstrate that vascular targeting of endothelial expression of glyocproteins is feasible to visualize the accumulation of near-IR fluorescent MECA-79 in lymph nodes, making this technology potentially useful to characterize processes of inflammation.
During endoscopy of the esophagus fluorescence images were recorded at a delay of 20 ns after pulsed laser excitation simultaneously with conventional reflected white light images. To label malignant cells (dysplasia, tumor) 5-aminolaevulinic acid was applied prior to fluorescence guided bi-opsy. In this way pre-malignant and malignant lesions were detected not seen previously during routine endoscopy.
Fluorescence images were recorded simultaneously with white light images to detect dyspasia or early malignancies during regular endoscopy of the upper gastrointestinal tract, after topical administration of 5-aminolaevulinic acid. Biopsies were taken at locations where fluorescence intensity were high compared with the mean fluorescence intensity of the image. Prompt and delayed fluorescence spectra of biopsies were subsequently recorded ex vivo, and normalized fluorescence intensities of Protoporphyrin IX derived from these spectra were compared with routine histology. In contrast to routine endoscopy, one early carcinoma and one signet-ring carcinoma were found in the stomach, and malignancies in a duodenal polyp. In addition, intestinal metaplasia could be visualized in the stomach of two patients, which had not been detected in biopsies taken prior to fluorescence endoscopy.
Many gastroenteropancreatic tumors express receptors for somatostatin (SST) and/or vasoactive intestinal peptide (VIP). These receptors can be used as molecular targets for the delivery of contrast agents for tumor diagnostics. We have synthesized conjugates consisting of a cyanine dye and an SST analogue or VIP for use as contrast agents in optical imaging. Receptor binding and internalization of these compounds were examined with optical methods in transfected RIN38 tumor cells expressing the SST2 receptor or a GFP- labeled VIP (VPAC1) receptor. Furthermore, biodistribution of the conjugates was examined by laser-induced fluorescence imaging in nude mice bearing SST2 or VPAC1 receptor- expressing tumors. After incubation of RIN38 SSTR2 cells in the presence of 100 nM indotricarbocyanine-SST analogue, cell-associated fluorescence increased, whereas no increase was observed when receptor-medicated endocytosis was inhibited. Indodicarbocyanine-VIP accumulated in RIN38 VPAC1 cells and co-localization with the GFP-labeled VPAC1 receptor was observed. After injection of indotricarbocyanine-SST analogue into tumor-bearing nude mice, SST2 receptor-positive tumors could be visualized for a time period from 10 min to at least 48 h. After application of indodicarbocyanine-VIP, a fluorescence signal in VIP1 receptor-expressing tumors was only detected during the first hour. We conclude that cyanine dye-labeled VIP and SST analogue are novel, targeted contrast agents for the optical imaging of tumors expressing the relevant receptor.
Many tumor cells are characterized by the overexpression of highly specific transferrin receptors. Transferrin is bound to its receptor, which is followed by rapid internalization into intracellular compartments; it can thus be used for the specific delivery of coupled contrast agents in to tumor cells. The aim of our study was to investigate the tumor- specificity of transferrin-modified contrast agents for optical imaging and magnetic resonance imaging (MRI). Indotricarbocyanine, a near-IR absorbing dye, and ultrasmall superparamagnetic iron oxide particles, a contrast material for MRI, were covalently coupled to transferrin and examined in vitro and in vivo for their potential as tumor-specific contrast agents. CEll culture experiments using HT29 and A431 tumor cells showed specific uptake of transferrin- modified contrast agents by the cells at 37 degrees C, while uptake was inhibited at 4 degrees C or in the presence of an excess of unlabeled transferrin. Optical imaging of tumor- bearing nude mice after intravenous injection of transferrin-ICC resulted in pronounced tumor fluorescence. Tumor specificity was also observe in MRI, where transferrin-USPIO accumulated in SMT2A tumor-bearing rats. We conclude that transferrin-modified contrast agents for optical imaging and MRI can be specifically delivered to tumor tissue by use of transferrin receptor pathways.
The influence of the molecular weight of cyanine dye- poly(ethylene glycol) (PEG) conjugates on their pharmacokinetic behavior and on the contrast between malignant and normal tissue in fluorescence images was studied. PEG conjugates with a molecular weight ranging from 1800 to 40000 g mol-1 were investigated in a rat model. A tunable, pulsed, solid-state laser system and an intensified CCD camera served to record fluorescence images of different tumor-bearing mice and rats. The time window of increased contrast between tumor and normal tissue in fluorescence images can be adjusted by the molecular weigh of PEG residues. Furthermore, we were able to demonstrate the visualization of subcutaneous blood vessels.
In this study indotricarbocyanines were investigated in vivo as near-infrared contrast agents. The known dye indocyanine green (ICG) has several disadvantages regarding its use in near-infrared imaging. ICG has a very short plasma half- life, limited tolerability and is unstable in aqueous solutions. Therefore, several indotricarbocyanine dyes, structurally related to ICG but with different hydrophilicities and physicochemical properties, were synthesized. The tolerability of synthesized dyes was tested in mice. The pharmacokinetic behavior and elimination characteristics were studied in a rat model. The in vivo imaging properties of synthesized dyes were investigated using a tunable, pulsed, solid state laser system for excitation and an intensified CCD camera for fluorescence imaging of different tumor-bearing nude mice models and mamma-carcinoma-bearing rat models. The dye-specific fluorescence exitance was followed at different times after dye administration. The results are demonstrated in comparison to indocyanine green. Synthesized hydrophilic indotricarbocyanine dyes had longer plasma half-lives and increasing renal elimination, corresponding to higher hydrophilicity. Tolerability in mice was increased up to 60- fold compared to ICG. Increased fluorescence exitance in tumors was observed for several dyes 24 h p.i. in the tumor models studied, whereas ICG showed no tumor fluorescence signal under the same conditions.
Fluorescence spectra of primary colon tumors, normal colonic tissue and lymph nodes were recorded ex vivo, following pulsed laser excitation at lambda equals 505 nm. Fluorescence was detected at zero delay and at a delay of 20 ns between the optical pulse and the opening of the intensified diode array detector. Generally, the spectra consist of a broad unspecific background (unspecific autofluorescence) and two characteristic fluorescence bands at lambda approximately equals 630 nm and lambda approximately equals 700 nm (specific autofluorescence). By delayed observation the intensity of the specific fluorescence diminishes less than the intensity of the unspecific background indicating a longer fluorescence decay time of the specific compared to the unspecific fluorescence. The fluorescence excitation and the fluorescence emission spectra of the specific autofluorescence as well as the fluorescence decay time are similar to those of porphyrins. Assuming that the relative amount of specific autofluorescence can be used to discriminate between primary tumors and normal colonic tissue or involved and non-involved lymph nodes, we have analyzed undelayed and delayed fluorescence spectra quantitatively to derive characteristic quantities for discrimination. We have investigated 18 primary colon tumors and associated normal colonic tissue as well as 174 lymph nodes. Out of 18 primary tumors classified by routine pathology we have correctly identified 16 by fluorescence analysis. Because of the rather weak specific autofluorescence in lymph nodes only 22 metastatically involved lymph nodes were detected out of 34 identified by routine pathology. Although the sensitivity of 65% is rather low a discrimination between involved and non-involved lymph nodes is of particular medical importance.
Images of superficially growing tumors marked with prophyrin-based photosensitizers (Photofrin II, P4P-mD) were recorded in vivo by delayed observation of fluorescence following pulsed laser excitation. A pulsed, solid state, widely tunable laser system was used together with an intensified CCD camera. The ratio of the fluorescence radiant exitances of tumor and normal tissue taken as contrast between tumor and surrounding tissue was studied systematically by varying excitation wavelength and delay time between the optical pulse and the opening of the intensifier of the CCD camera. The dependence of this ratio on excitation wavelength was explained qualitatively using a simple one-dimentional mathematical model, taking the penetration depth of the exciting laser radiation and the thickness of the tumor and the layer of normal tissue above it into account. Besides fluorescence images, delayed fluorescence emission spectra of tumor and normal tissue were recorded in vivo.
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