Validation of Fluorescence Molecular Tomography/Micro-CT Multimodal Imaging In Vivo in Rats

Purpose

Rats are important preclinical models for studying breast cancer metastasis and bone pathologies. In these research areas, fluorescence molecular tomography (FMT) is commonly applied for quantitative three-dimensional (3D) imaging in mice. However, uncertainties due to strong depth dependency of FMT signal and spatial resolution require a validation study in rats.

Procedure

FMT performance in rats was assessed based on co-registered FMT/micro-computed tomography (micro-CT) reconstructed volumes obtained from optical phantoms and from models relevant for tumor imaging, bone remodeling and biodistribution analysis of nanoparticles.

Results

FMT reconstructions within 20-mm-thick optical phantoms were accurate (95?±?11 % recovery), precise (CV?≤?8 %) and linear (R ²?>?0.9788) over a range of 78–2,500 nM of the near infrared fluorescent agent VivoTag 750 (VT₇₅₀). In vivo, implanted defined fluorescent targets yielded a recovery of 105?±?5 % and successfully co-registered with micro-CT delineated structures. Additionally, using the bone-targeting imaging agent Osteosense 750, regions of neo bone formation identified by FMT could be mapped to the region of epiphyseal growth plates observed in micro-CT images. Finally, as a proof of concept, to monitor nanoparticulate drug pharmacokinetics in rat subjects the accumulation/clearance of VT₇₅₀–albumin conjugate in/from the liver was followed at 11 different time points over a period of 2 weeks by FMT/micro-CT.

Conclusions

FMT imaging has been validated in optical phantoms as well as in 160 g rats, and sequential FMT/micro-CT imaging can be considered as a useful tool for preclinical research in rats.     

血管内超声对粥样硬化冠状动脉成像的研究

研究目的在于评价血管内超声（ＩＶＵＳ）观察粥样硬化冠状动脉（ＣＡ）的安全性和可行性。利用３．５Ｆ，３０ＭＨｚ超声导管对１１例冠心病患者的２０支冠状动脉节段进行了检查，所有病人均顺利接ＩＶＵＳ检查，５例血管造影提示冠脉左主干正常的血管段，ＩＶＵＳ显示有内膜轻度增厚或局灶性斑块，１５支血管造影提示ＣＡ管腔狭窄的血管段，ＩＶＵＳ发现有中至重度的内膜增厚，操作中未发现严重并发症。结论：血管内超声检查是安全可行的，它可提供异常ＣＡ管壁形态学的详细信息。     

Characterization and In Vitro and In Vivo Testing of CB2-Receptor- and NGAL-Targeted Paramagnetic Micelles for Molecular MRI of Vulnerable Atherosclerotic Plaque

Purpose

Atherosclerotic plaque macrophages express the peripheral cannabinoid receptor (CB2-R) and promote fibrous cap degradation by secretion of neutrophil gelatinase-associated lipocalin 2 (NGAL). In this study, we report the preparation, characterization, and in vitro and in vivo testing of double-labeled (MR and fluorescent) CB2-R- and NGAL-targeted micelles.

Procedures/Results

Specific CB2-R agonists or antibodies directed to 24p3 (mouse homolog of NGAL) were incorporated into di-oleoyl-polyethylene glycol-phosphatidylethanolamine 1000 (DOPE-PEG1000) micelles or di-stearoyl-polyethylene glycol-phosphatidylethanolamine 2000 (DSPE-PEG2000) micelles. The hydrodynamic diameter, determined by dynamic light scattering, was 16.5 and 19.0 nm for CB2-R-targeted DOPE-PEG1000 and DSPE-PEG2000 micelles, respectively, and 23.0 nm for Ab-conjugated DSPE-PEG2000 micelles. In vitro and in vivo MRI and fluorescence microscopy showed specific binding of CB2-R-targeted and 24p3-targeted micelles to in vitro systems and to aortic plaque in apoE^?/?/eNOS^?/? mice, respectively.

Conclusions

CB2-R- and NGAL-targeted micelles show promise as tools for in vivo characterization of vulnerable plaque.     

In Vivo pH Imaging with 99mTc-pHLIP

Purpose

A novel molecular imaging agent has been developed recently, which stains tissues of low extracellular pH [pH (low) insertion peptide, pHLIP^?]. A pH-dependent process of peptide folding and insertion into cell membranes has been found in vitro. Targeting of acidic solid tumours has been demonstrated in vivo using fluorescence and PET labels. Here, we present proof of feasibility studies of pHLIP with a single-photon emission computed tomography (SPECT) label, ^99mTc-AH114567, with focus on preclinical efficacy and imageability.

Procedures

Lewis lung carcinoma, lymph node carcinoma of the prostate and prostate adenocarcinoma tumour xenografts were grown in mice and characterised by the angiogenesis marker ^99mTc-NC100692 and by extracellular pH measurements with ³¹P-MRS of 3-aminopropyl phosphonate. Biodistribution was assessed and CT/SPECT imaging performed. Oral administration of bicarbonate served as control.

Results and Conclusion

Tc-AH114567 can be obtained via a robust synthesis with good radiolabelling profile and improved formulation. The tracer retains the pH-dependent ability to insert into membranes and to target tumours with similar pharmacokinetics and efficacy that had been demonstrated earlier for pHLIP with optical or ⁶⁴Cu PET labels. Despite the inherent challenges of SPECT compared to optical and PET imaging, e.g., in terms of lower sensitivity, ^99mTc-AH114567 shows adequate image quality and contrast. The main development need for transitioning SPECT labelled pHLIP into the clinic is more rapid background signal reduction, which will be the focus of a subsequent optimisation study.     

In Vivo Molecular Imaging Using Low-Boiling-Point Phase-Change Contrast Agents: A Proof of Concept Study

Sub-micron phase-change contrast agents (PCCAs) have been proposed as a tool for ultrasound molecular imaging based on their potential to extravasate and target extravascular markers and also because of the potential to image these contrast agents with a high contrast-to-tissue ratio. We compare in vivo ultrasound molecular imaging with targeted low-boiling-point PCCAs and targeted microbubble contrast agents. Both agents were targeted to the intravascular (endothelial) integrin α_vß₃via a cyclic RGD peptide (cyclo-Arg–Gly–Asp–D-Tyr–Cys) mechanism and imaged in vivo in a rodent fibrosarcoma model, which exhibits angiogenic microvasculature. Signal intensity was measured using two different techniques, conventional contrast-specific imaging (amplitude/phase modulation) and a droplet vaporization imaging sequence, which detects the unique signature of vaporizing PCCAs. Data indicate that PCCA-specific imaging is more sensitive to small numbers of bound agents than conventional contrast imaging. However, data also revealed that contrast from targeted microbubbles was greater than that provided by PCCAs. Both control and targeted PCCAs were observed to be retained in tissue post-vaporization, which was expected for targeted agents but not expected for control agents. The exact mechanism underlying this observation remains unknown.     

重组人肿瘤坏死因子-α体内外对HL-60细胞作用的研究

为了研究重组人肿瘤坏死因子-α（recombinant human tumor necrosis factor-alpha,rhTNF-α）在体外及体内对人髓系白血病细胞株HL-60细胞的作用,应用MTT法和集落形成试验测定HL-60细胞的增殖抑制,采用AO/EB荧光染色法、流式细胞术和TdT酶介导的缺口末端标记（TUNEL）法检测凋亡细胞;利用HL-60细胞裸鼠异种移植瘤模型观察给药后瘤体重量、组织病理的改变,并采用透射电镜以及TUNEL法检测瘤组织细胞的凋亡.结果表明：rhTNF-α对HL-60细胞的增殖具有明显的抑制作用,呈时间和浓度依赖性;AO/EB染色后可见rhTNF-α处理组HL-60细胞核内染色质浓缩聚集或成碎片,呈现凋亡表象;流式细胞术显示随着rhTNF-α浓度的增加,细胞凋亡阳性率逐渐增高;TUNEL法检测显示,3 200 U/ml rhTNF-α作用于HL-60细胞48小时凋亡阳性率可达37.5%.在体内,rhTNF-α可抑制HL-60细胞裸鼠异种移植瘤生长,抑制率最高可达60.33%;病理检查发现,rhTNF-α处理组的瘤组织有程度不等的出血坏死区域;透射电镜及TUNEL法检测表明,处理组的瘤组织可见有较多凋亡细胞.结论：rhTNF-α在体内、外均可抑制HL-60细胞增殖,并诱导其凋亡.     

Quantitative Ultrasound Imaging: In Vivo Results in Normal Liver

A method for quantitative imaging of ultrasonic backscatter levels has been implemented on a clinical imager. The method is based on comparing echo signal data from a sample or patient to echo data processed in the same way but acquired from a reference phantom. The attenuation coefficient and the backscatter coefficient of the reference phantom are known, permitting these quantities to be estimated for the sample. In the present paper, the spatial location of echo data acquisition is retained in the backscatter data analysis; quantitative "backscatter estimator" images are constructed, from which the backscatter coefficient over a region of interest may be obtained. When applied to human liver images, backscatter coefficients determined in 10 normal subjects were in approximate agreement with in vitro liver backscatter coefficients reported by previous workers.     

Synthesis and Evaluation of Diindole-Based MRI Contrast Agent for In Vivo Visualization of Necrosis

Purpose

Noninvasive imaging of cell necrosis can provide an early evaluation of tumor response to treatments. Here, we aimed to design and synthesize a novel diindole-based magnetic resonance imaging (MRI) contrast agent (Gd-bis-DOTA-diindolylmethane, Gd-DIM) for assessment of tumor response to therapy at an early stage.

Procedures

The oil-water partition coefficient (Log P) and relaxivity of Gd-DIM were determined in vitro. Then, its necrosis avidity was examined in necrotic cells in vitro and in rat models with microwave ablation-induced muscle necrosis (MAMN) and ischemia reperfusion-induced liver necrosis (IRLN) by MRI. Visualization of tumor necrosis induced by combretastatin A-4 disodium phosphate (CA4P) was evaluated in rats bearing W256 orthotopic liver tumor by MRI. Finally, DNA binding assay was performed to explore the possible necrosis-avidity mechanism of Gd-DIM.

Results

The Log P value and T1 relaxivity of Gd-DIM is ??2.15?±?0.01 and 6.61 mM^?1 s^?1, respectively. Gd-DIM showed predominant necrosis avidity in vitro and in vivo. Clear visualization of the tumor necrosis induced by CA4P was achieved at 60 min after administration of Gd-DIM. DNA binding study indicated that the necrosis-avidity mechanism of Gd-DIM may be due to its binding to exposed DNA in necrotic cells.

Conclusion

Gd-DIM may serve as a promising necrosis-avid MRI contrast agent for early assessment of tumor response to therapy.

     

Introduction: feature issue on In Vivo Microcirculation Imaging

The editors introduce the Biomedical Optics Express feature issue, "In Vivo Microcirculation Imaging," which includes 14 contributions from the biomedical optics community, covering such imaging techniques as optical coherence tomography, photoacoustic microscopy, laser Doppler /speckle imaging, and near infrared spectroscopy and fluorescence imaging.     

Imaging Cobalamin Uptake within Malignant Breast Tumors In Vivo

Molecular Imaging and Biology - To image the uptake of cobalamin (Cbl) within malignant breast tumors in vivo. Prior to surgery 20 female patients with clinically suspected breast tumors were...     

Comparison of Monkeypox Viruses Pathogenesis in Mice by In Vivo Imaging

Monkeypox viruses (MPXV) cause human monkeypox, a zoonotic smallpox-like disease endemic to Africa, and are of worldwide public health and biodefense concern. Using viruses from the Congo (MPXV-2003-Congo-358) and West African (MPXV-2003-USA-044) clades, we constructed recombinant viruses that express the luciferase gene (MPXV-Congo/Luc+and MPXV-USA-Luc+) and compared their viral infection in mice by biophotonic imaging. BALB/c mice became infected by both MPXV clades, but they recovered and cleared the infection within 10 days post-infection (PI). However, infection in severe combined immune deficient (SCID) BALB/c mice resulted in 100% lethality. Intraperitoneal (IP) injection of both MPXV-Congo and MPXV-Congo/Luc+resulted in a systemic clinical disease and the same mean time-to-death at 9 (±0) days post-infection. Likewise, IP injection of SCID-BALB/c mice with MPXV-USA or the MPXV-USA-Luc+, resulted in similar disease but longer (P<0.05) mean time-to-death (11±0 days) for both viruses compared to the Congo strains. Imaging studies in SCID mice showed luminescence in the abdomen within 24 hours PI with subsequent spread elsewhere. Animals infected with the MPXV-USA/Luc+had less intense luminescence in tissues than those inoculated with MPXV-Congo/Luc+, and systemic spread of the MPXV-USA/Luc+virus occurred approximately two days later than the MPXV-Congo/Luc+. The ovary was an important target for viral replication as evidenced by the high viral titers and immunohistochemistry. These studies demonstrate the suitability of a mouse model and biophotonic imaging to compare the disease progression and tissue tropism of MPX viruses.     

In Vivo Imaging of miR-221 Biogenesis in Papillary Thyroid Carcinoma

Purpose  To investigate the overexpression of miR-221 in papillary thyroid carcinoma (PTC), we developed a Gaussia luciferase (Gluc) system regulated by miR-221. Procedures  Quantities of primary or mature miR-221 in normal thyroid cells (HT-ori3) and in PTC (NPA, TPC-1) were measured by quantitative real-time polymerase chain reaction. Cytomegalovirus (CMV)/Gluc-3xPT_miR221, which included three perfect complementary target sequences repeats of miR221 in the 3′-untranslated region of Gluc, was transfected into cells with pre-miR-221 or anti-miR-221 and Gluc activities were then compared in vitro and in vivo. Results  Primary or mature miR-221 were overexpressed in PTC as compared with HT-ori3. In cells transfected with the Gaussia luciferase reporter system (CMV/Gluc-3xPT_miR221), Gluc activities were regulated according to miR-221 levels in vitro and in vivo. Conclusions  These results suggest that the devised CMV/Gluc-3xPT_miR221 system may be a useful tool for monitoring quantities of endogenous miR-221 in cells or living organisms.     

Imaging of Protein Synthesis: In Vitro and In Vivo Evaluation of 44Sc-DOTA-Puromycin

Purpose

The purpose of this study was to investigate whether ⁴⁴Sc-labeled puromycin can be utilized for imaging of protein synthesis in vivo.

Methods

For micro-positron emission tomographic (μPET) studies, 20–25 MBq of [⁴⁴Sc]-DOTA-puromycin was administered to tumor-bearing rats, and animals were scanned for 1 h dynamically. Results were further validated by dissecting organs and tissues of the animals after the measurement and in vitro blocking experiments using puromycin or cycloheximide to block protein synthesis.

Results

μPET images of tumor-bearing rats showed significant tumor uptake of [⁴⁴Sc]-DOTA-puromycin and a clear-cut tumor visualization. In both blocking experiments, cellular uptake of [⁴⁴Sc]-DOTA-puromycin ([⁴⁴Sc]-DOTA-Pur) could be suppressed by blocking protein synthesis.

Conclusions

We report for the first time successful μPET imaging with ⁴⁴Sc obtained from a ⁴⁴Ti/⁴⁴Sc generator, as well as noninvasive μPET imaging of ribosomal activity, respectively protein synthesis, with a puromycin-based radiopharmaceutical and the direct correlation between cellular uptake of [⁴⁴Sc]-DOTA-Pur and protein synthesis.     

Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.     

In Vivo Magnetic Resonance Imaging of Transgenic Mice Expressing Human Ferritin

Objective

This study aims to produce the transgenic mice (TG) engineered for magnetic resonance imaging (MRI) studies based on the ubiquitous expression of ferritin MRI reporter gene in diverse tissues.

Procedures

Transgenic mice (TG) expressing myc-tagged human heavy chain ferritin (myc-hFTH) under the control of a ubiquitous CAG promoter were produced. The expression of myc-hFTH in diverse tissues of the myc-hFTH TG was assessed by RT-PCR, Western blotting, and immunohistochemistry. The iron accumulation and the deposition of ferritin aggregates in tissues of myc-hFTH TG and WT were analyzed by Prussian blue staining and transmission electron microscopy. The myc-hFTH TG (n?=?9) and wild-type mice (WT) (n?=?4) were subjected to MRI on 9.4 T MR scanner. An eight-point T ₂ ^* mapping was performed using a multiple gradient echo sequence, and T ₂ ^* value was estimated pixel by pixel by using a routine least-squares fitting algorithm.

Results

We generated the myc-hFTH TG expressing myc-hFTH in brain, heart, liver, lung, spleen, pancreas, kidney, and intestine. The myc-hFTH TG showed no apparent pathological symptoms and no histological changes compared to WT. The expression of myc-hFTH in the brain and liver tissues of myc-hFTH TG led to a significant decrease in T₂* values, as shown by noninvasive MRI, compared to WT (P?<?0.05, TG vs. WT).

Conclusions

This study demonstrates that the novel myc-hFTH TG, which expresses an MRI reporter in many tissues, would be a valuable animal model of FTH-based molecular imaging in which to study potential therapies for cell and tissue grafting using an MRI technique. These mice could also serve to study disease related with iron metabolism.     

In Vivo Imaging of Microvasculature during Anesthesia with High-Resolution Photoacoustic Microscopy

Anesthesia monitoring is extremely important in improving the quality of anesthesia and ensuring the safety of patients in operation. Photoacoustic microscopy (PAM) is proposed to in vivo image the skin microvasculature of 10 nude mice undergoing general anesthesia by using the isoflurane gas with a concentration of 3%. Benefiting from strong optical absorption of hemoglobin, PAM has good contrast and high resolution in mapping of microvasculature. A series of high quality images can clearly reveal the subtle changes of capillaries in morphology over time. Two indices, vessel intensity and vessel density, are extracted from these images to measure the microvasculature quantitatively. The imaging results show that the vessel intensity and density are increased over time. After 65?min, the vessel intensity increased 42.7?±?8.6% and the density increased 28.6?±?12.2%. These indices extracted from photoacoustic images accurately reflect the greater blood perfusion undergoing general anesthesia. Additionally, abnormal reductions of vessel intensity and density are also observed as overtime anesthesia. This preclinical study suggests that PAM holds potential to monitor anesthesia by imaging the skin microvasculature.