活体动物体内光学成像技术的研究进展及其应用

活体动物体内光学成像是利用基因改构进行内源性成像试剂或外源性成像试剂标记细胞、蛋白或DNA，从而非侵入性地报告小动物体内的特定生物学事件的技术。活体成像可以直观灵敏地监测基因的表达模式、标记和示踪细胞、探讨蛋白间的相互作用，因而这一技术被广泛地用于分析基因的表达模式、评价基因治疗效果、评估肿瘤的发生和转移、监测移植器官等。简要综述了现有活体动物体内光学成像技术的基本原理、技术进展和相关应用。     

In Vivo Imaging of Human Cerebral Acetylcholinesterase

Abstract: We report here the first positron emission tomography (PET) images showing the in vivo regional distribution of acetylcholinesterase (AChE) in human brain. The study was carried out in eight healthy human volunteers using as a tracer [¹¹C]physostigmine ([¹¹C]PHY), an inhibitor of AChE. After intravenous injection of [¹¹C]PHY, radioactivity was rapidly taken up in brain tissue and reached maximal uptake within a few minutes, following a regional pattern mostly related to cerebral perfusion. After the peak, the cerebral radioactivity gradually decreased with a half-life varying from 20 to 35 min, depending on the brain structure. [¹¹C]PHY retention was higher in regions rich in AChE, such as the striatum (half-life, 35 min), than in regions poor in AChE, such as the cerebral cortex (half-life, 20 min). At later times (25–35 min postinjection), the cerebral distribution of [¹¹C]PHY was typical of AChE activity: putamen-caudate > cerebellum > brainstem > thalamus > cerebral cortex, with a striatal to cortex ratio of 2. These results suggest that PET studies with [¹¹C]PHY can provide in vivo brain mapping of human AChE and are promising for the study of changes in AChE levels associated with neurodegenerative diseases.     

小动物活体成像技术的应用

小动物活体成像技术在国内外得到越来越多的普及应用,极大地促进了生命科学特别是肿瘤研究的发展。本文就小动物活体成像技术的原理、标记方法和实际应用做简单介绍。     

In Vivo Detection of Extrapancreatic Insulin Gene Expression in Diabetic Mice by Bioluminescence Imaging

Background

Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP).

Methods

The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis.

Results

Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia.

Conclusions

BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.     

在体生物发光成像技术在生物医学中的应用

在体生物发光成像技术采用萤光素酶基因标记细胞及DNA,利用灵敏的光学检测仪器,实时直观地监测活体小动物体内感染性疾病的发生发展、肿瘤的生长及转移及引发的免疫反应、特定基因的表达等诸多生物过程。通过对同一组实验对象在不同时间点进行记录,跟踪同一观察目标(标记细胞、病原微生物或基因)的移动及变化,与传统的动物实验方法相比,在体生物发光成像得到的数据具有更高的可信度。近年来因其灵敏度较高、不涉及放射性物质、所得结果直观等优势,该技术已普遍应用于生物医学、药物开发等研究领域。     

Mechanical Stress Is a Pro-Inflammatory Stimulus in the Gut: In Vitro,In Vivo and Ex Vivo Evidence

Aims

Inflammatory infiltrates and pro-inflammatory mediators are found increased in obstructive and functional bowel disorders, in which lumen distention is present. However, what caused the low level inflammation is not well known. We tested the hypothesis that lumen distention- associated mechanical stress may induce expression of specific inflammatory mediators in gut smooth muscle.

Methods

Static mechanical stretch (18% elongation) was applied in vitro in primary culture of rat colonic circular smooth muscle cells (RCCSMCs) with a Flexercell FX-4000 Tension Plus System. Mechanical distention in vivo was induced in rats with an obstruction band placed in the distal colon.

Results

In the primary culture of RCCSMCs, we found that static stretch significantly induced mRNA expression of iNOS, IL-6, and MCP-1 in 3 hours by 6.0(±1.4), 2.5(±0.5), and 2.2(±0.5) fold (n = 6∼8, p<0.05), respectively. However, gene expression of TNF-α, IL-1β, and IL-8 was not significantly affected by mechanical stretch. In the in vivo model of colon obstruction, we found that gene expression of iNOS, IL-6, and MCP-1 is also significantly increased in a time-dependent manner in the mechanically distended proximal segment, but not in the sham controls or distal segments. The conditioned medium from the muscle strips of the stretched proximal segment, but not the distal segment or control, significantly induced translocation and phosphorylation of NF-κB p65. This treatment further increased mRNA expression of inflammatory mediators in the naïve cells. However, treatment of the conditioned medium from the proximal segment with neutralizing antibody against rat IL-6 significantly attenuated the activation of NF-κB and gene expression of inflammatory mediators.

Conclusions

Our studies demonstrate that mechanical stress induces gene expression of inflammatory mediators i.e. iNOS, IL-6, and MCP-1 in colonic SMC. Further ex vivo study showed that mechanical stress functions as a pro-inflammatory stimulus in the gut.     

In Vivo Imaging and Characterization of Actin Microridges

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.     

Making the Brain Glow: In Vivo Bioluminescence Imaging to Study Neurodegeneration

Bioluminescence imaging (BLI) takes advantage of the light-emitting properties of luciferase enzymes, which produce light upon oxidizing a substrate (i.e., d-luciferin) in the presence of molecular oxygen and energy. Photons emitted from living tissues can be detected and quantified by a highly sensitive charge-coupled device camera, enabling the investigator to noninvasively analyze the dynamics of biomolecular reactions in a variety of living model organisms such as transgenic mice. BLI has been used extensively in cancer research, cell transplantation, and for monitoring of infectious diseases, but only recently experimental models have been designed to study processes and pathways in neurological disorders such as Alzheimer disease, Parkinson disease, or amyotrophic lateral sclerosis. In this review, we highlight recent applications of BLI in neuroscience, including transgene expression in the brain, longitudinal studies of neuroinflammatory responses to neurodegeneration and injury, and in vivo imaging studies of neurogenesis and mitochondrial toxicity. Finally, we highlight some new developments of BLI compounds and luciferase substrates with promising potential for in vivo studies of neurological dysfunctions.     

Peptide-Based Optical uPAR Imaging for Surgery: In Vivo Testing of ICG-Glu-Glu-AE105

Near infrared intra-operative optical imaging is an emerging technique with clear implications for improved cancer surgery by enabling a more distinct delineation of the tumor margins during resection. This modality has the potential to increase the number of patients having a curative radical tumor resection. In the present study, a new uPAR-targeted fluorescent probe was developed and the in vivo applicability was evaluated in a human xenograft mouse model. Most human carcinomas express high level of uPAR in the tumor-stromal interface of invasive lesions and uPAR is therefore considered an ideal target for intra-operative imaging. Conjugation of the flourophor indocyanine green (ICG) to the uPAR agonist (AE105) provides an optical imaging ligand with sufficiently high receptor affinity to allow for a specific receptor targeting in vivo. For in vivo testing, human glioblastoma xenograft mice were subjected to optical imaging after i.v. injection of ICG-AE105, which provided an optimal contrast in the time window 6–24 h post injection. Specificity of the uPAR-targeting probe ICG-AE105 was demonstrated in vivo by 1) no uptake of unconjugated ICG after 15 hours, 2) inhibition of ICG-AE105 tumor uptake by a bolus injection of the natural uPAR ligand pro-uPA, and finally 3) the histological colocalization of ICG-AE105 fluorescence and immunohistochemical detected human uPAR on resected tumor slides. Taken together, our data supports the potential use of this probe for intra-operative optical guidance in cancer surgery to ensure complete removal of tumors while preserving adjacent, healthy tissue.     

Two-dimensional Fast Surface Imaging Using a Handheld Optical Device: In Vitro and In Vivo Fluorescence Studies

Near-infrared (NIR) optical imaging is a noninvasive and nonionizing modality that is emerging as a diagnostic tool for breast cancer. The handheld optical devices developed to date using the NIR technology are predominantly developed for spectroscopic applications. A novel handheld probe-based optical imaging device has been recently developed toward area imaging and tomography applications. The three-dimensional (3D) tomographic imaging capabilities of the device have been demonstrated from previous fluorescence studies on tissue phantoms. In the current work, fluorescence imaging studies are performed on tissue phantoms, in vitro, and in vivo tissue models to demonstrate the fast two-dimensional (2D) surface imaging capabilities of this flexible handheld-based optical imaging device, toward clinical breast imaging studies. Preliminary experiments were performed using target(s) of varying volume (0.23 and 0.45 cm³) and depth (1–2 cm), using indocyanine green as the fluorescence contrast agent in liquid phantom, in vitro, and in vivo tissue models. The feasibility of fast 2D surface imaging (∼5 seconds) over large surface areas of 36 cm² was demonstrated from various tissue models. The surface images could differentiate the target(s) from the background, allowing a rough estimate of the target''s location before extensive 3D tomographic analysis (future studies).     

Neutralization of Schwann Cell-Secreted VEGF Is Protective to In Vitro and In Vivo Experimental Diabetic Neuropathy

The pathogenetic role of vascular endothelial growth factor (VEGF) in long-term retinal and kidney complications of diabetes has been demonstrated. Conversely, little is known in diabetic neuropathy. We examined the modulation of VEGF pathway at mRNA and protein level on dorsal root ganglion (DRG) neurons and Schwann cells (SC) induced by hyperglycaemia. Moreover, we studied the effects of VEGF neutralization on hyperglycemic DRG neurons and streptozotocin-induced diabetic neuropathy. Our findings demonstrated that DRG neurons were not affected by the direct exposition to hyperglycaemia, whereas showed an impairment of neurite outgrowth ability when exposed to the medium of SC cultured in hyperglycaemia. This was mediated by an altered regulation of VEGF and FLT-1 receptors. Hyperglycaemia increased VEGF and FLT-1 mRNA without changing their intracellular protein levels in DRG neurons, decreased intracellular and secreted protein levels without changing mRNA level in SC, while reduced the expression of the soluble receptor sFLT-1 both in DRG neurons and SC. Bevacizumab, a molecule that inhibits VEGF activity preventing the interaction with its receptors, restored neurite outgrowth and normalized FLT-1 mRNA and protein levels in co-cultures. In diabetic rats, it both prevented and restored nerve conduction velocity and nociceptive thresholds. We demonstrated that hyperglycaemia early affected neurite outgrowth through the impairment of SC-derived VEGF/FLT-1 signaling and that the neutralization of SC-secreted VEGF was protective both in vitro and in vivo models of diabetic neuropathy.     

Evidence of In Vivo Prophage Induction during Clostridium difficile Infection

Prophages contribute to the evolution and virulence of most bacterial pathogens, but their role in Clostridium difficile is unclear. Here we describe the isolation of four Myoviridae phages, ϕMMP01, ϕMMP02, ϕMMP03, and ϕMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering from C. difficile infection (CDI). Furthermore, identical prophages were found in the chromosomes of C. difficile isolated from the corresponding fecal samples. We therefore provide, for the first time, evidence of in vivo prophage induction during CDI. We completely sequenced the genomes of ϕMMP02 and ϕMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ϕMMP04. We also studied the mobility of ϕMMP02 and ϕMMP04 prophages in vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ϕMMP04. In summary, our study highlights the extensive genetic diversity and mobility of C. difficile prophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobility in vitro and probably in vivo as well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution of C. difficile.     

Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas

The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of ^99mTc-MAG-aptamer and ¹¹¹In-DOTA-F3B. ^99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of ¹¹¹In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for ¹¹¹In-DOTA-F3B with 2.0%ID/g than for the ¹¹¹In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.