High-Resolution Intravital Imaging for Monitoring the Transplanted Islets in Mice

Intravital imaging techniques will be a valuable tool to monitor the post-transplantation dynamics of the cells/tissues in regenerative medicine research. Among the conventional live imaging techniques, the cranial window model has various advantages regarding resolution, longevity, and easy manipulability. We describe the use of the cranial window model to visualize the post-transplantation processes of primary pancreatic islets in the living mouse. Macroscopic or microscopic analyses were performed to evaluate the post-transplantation dynamics of primary murine islets, including the revascularization process inside the cranium. Consistent with earlier literature on clinical outcomes of islet transplantation, marked loss of transplanted islets was observed within 7 days. Intravital confocal microscope analysis revealed that functional revascularization seldom occurred in the central regions of the transplants. Our results suggest that the cranial window model offers an ideal platform for understanding cellular dynamics, through the possibility of long-term imaging studies over time scales. This platform is possibly applied not only for transplant studies of pancreatic islets, but also for other endodermal cell/tissue types in vivo.     

Computer-generated three-dimensional animation of the mitral valve

OBJECTIVE: Three-dimensional motion-capture data offer insight into the mechanical differences of mitral valve function in pathologic states. Although this technique is precise, the resulting time-varying data sets can be both difficult to interpret and visualize. We used a new technique to transform these 3-dimensional ovine numeric analyses into an animated human model of the mitral apparatus that can be deformed into various pathologic states. METHODS: In vivo, high-speed, biplane cinefluoroscopic images of tagged ovine mitral apparatus were previously analyzed under normal and pathologic conditions. These studies produced serial 3-dimensional coordinates. By using commercial animation and custom software, animated 3-dimensional models were constructed of the mitral annulus, leaflets, and subvalvular apparatus. The motion data were overlaid onto a detailed model of the human heart, resulting in a dynamic reconstruction. RESULTS: Numeric motion-capture data were successfully converted into animated 3-dimensional models of the mitral valve. Structures of interest can be isolated by eliminating adjacent anatomy. The normal and pathophysiologic dynamics of the mitral valve complex can be viewed from any perspective. CONCLUSION: This technique provides easy and understandable visualization of the complex and time-varying motion of the mitral apparatus. This technology creates a valuable research and teaching tool for the conceptualization of mitral valve dysfunction and the principles of repair.     

Contrast specific imaging in the detection and localization of prostate cancer

Prostate cancer (PCa) is the most common cancer in elderly men and is one of the most important causes of death from cancer in men. The diagnosis of PCa is based on a combination of digital rectal examination, PSA and transrectal ultrasound (TRUS). However, this combination does not reach the accuracy of detection and localization necessary for proper decisions on treatment methods. Therefore, biopsies are performed in all cases for which the suspicion of PCa is raised. Even with biopsies, staging and grading of PCa is far from optimal. More accurate imaging is necessary to improve the biopsy sampling, the goals being to replace systematic biopsies by a targeted approach and to improve staging and grading of PCa. Ultrasound imaging of the prostate remains the first choice of imaging to visualize the prostate, however, gray-scale ultrasound imaging has an accuracy of about 50–60% for the detection of PCa and TRUS used for local staging has an even lower accuracy. The development of PCa is associated with changes in the metabolism of tumor cells, and therefore with changes in the blood perfusion of the involved tissue. This paper focuses on contrast specific imaging techniques to visualize these changes in blood perfusion. Techniques such as color and power Doppler imaging, and contrast enhanced imaging techniques using color and power Doppler, harmonic imaging and intermittent imaging are discussed.     

Medical applications of diffraction enhanced imaging

We have developed a new X-ray imaging technique, diffraction enhanced imaging (DEI), which can be used to independently visualize the refraction and absorption of an object. The images are almost completely scatter-free, allowing enhanced contrast of objects that develop small angle scattering. The combination of these properties has resulted in images of mammography phantoms and tissues that have dramatically improved contrast over standard imaging techniques. This technique potentially is applicable to mammography and other fields of medical X-ray imaging and to radiology in general, as well as possible use in nondestructive testing and X-ray computed tomography. Images of various tissues and materials are presented to demonstrate the wide applicability of this technique to medical and biological imaging.     

Effect of pentosan, a novel cancer chemotherapeutic agent, on prostate cancer cell growth and motility.

Pentosan is a new chemotherapeutic drug which is currently in Phase I clinical trials. In our experimental systems, in vivo, pentosan inhibits the growth of the highly metastatic MAT-LyLu (MLL) Dunning R3327 prostate cancer cell line only at toxic doses and has no apparent effect on growth in vitro. The mechanism of tumor inhibition of this drug is unknown; however, in vitro, pentosan exhibits a potent inhibition of cell motility. Cell motility is essential for tumor cell metastasis and angiogenesis. By blocking cell motility, pentosan has the potential to inhibit both tumor growth and metastasis. We have characterized the mechanism of motility inhibition by pentosan and believe it alters cell-extracellular matrix interactions. The mechanism of motility inhibition by pentosan appears to be independent of cytoskeletal structural alterations, including changes in microfilament and microtubule networks. Pentosan acts through a different mechanism than suramin, a drug which inhibits motility through inhibition of growth factor effects. In vitro, pentosan alters cellular contacts with the extravascular matrix and inhibits cell motility. In vivo, pentosan prolongs survival of rats injected with MLL cells by 25%, but did not appear to decrease the rate of primary tumor growth or the number of metastatic lesions in the treated animals. These data suggest that, in vivo, pentosan acts through an as yet undefined mechanism.     

Bildgebung des Nierenzellkarzinoms

Renal cell cancer is the most frequently encountered malignant tumor of the kidney. The modern imaging techniques computed tomography (CT) and magnetic resonance imaging (MRI) play a vital role in the diagnostic work-up of renal masses. Contrast-enhanced ultrasound (CEUS) is able to visualize subtle enhancement in hypovascular tumors with high sensitivity. Recent advances in multidetector CT (MDCT) enable high resolution imaging of local findings. CT is able to provide large volume coverage of the chest and abdomen within seconds for the assessment of lymph node and distant metastasis staging. Local renal cell cancer staging can be achieved with similar diagnostic accuracy using CT or MRI. Both modalities allow visualization of a tumor’s relationship to the renal collecting system and pelvis, the renal artery and veins including the IVC. It is the radiologist’s task to accurately detect and stage renal malignances in order that each individual patient receives the therapy best suited to them.     

一种可用活体荧光成像技术检测的前列腺原位肿瘤模型的建立

目的建立可用活体荧光成像技术动态观察前列腺原位肿瘤发生及生长的裸鼠模型。方法 10只BALB/c裸鼠前列腺背侧包膜内注入携带红色荧光蛋白（RFP）的前列腺癌PC-3细胞（PR7细胞）悬液20μL,第2、4、6、8周分别用非侵入式活体分子影像系统观察前列腺原位肿瘤发生及其转移情况。尸检取前列腺原位肿瘤、肺、肝、肾、股骨、盆腔淋巴结等组织,制成冰冻切片,通过荧光显微镜下观察及HE染色等方法分别检测肿瘤发生及有无转移等情况。结果第2周开始可用非侵入式活体分子影像系统检测到前列腺原位肿瘤形成,第4周开始可于裸鼠下腹部触及原位肿瘤,第6～8周裸鼠逐渐出现恶液质。荧光信号随原位肿瘤长大而增强,但并未见明显转移灶。尸检也未发现转移灶。结论前列腺包膜内注入携带RFP的前列腺癌PR7细胞可成功建立可用活体荧光成像技术监测的前列腺原位肿瘤模型,该模型可作为研究前列腺肿瘤发生发展及治疗方式的较理想的动物模型。     

人胃癌原位移植转移动物模型的构建

目的构建人胃癌原位移植转移动物模型,并利用活体成像系统对肿瘤转移进行可视化的实时监测评价。 方法构建带有绿色荧光蛋白序列和萤火虫荧光素酶序列的慢病毒重组表达质粒;慢病毒包装感染人BGC-823胃癌细胞株,筛选得到BGC-823-eGFP-luc2稳转株;细胞接种高度免疫缺陷小鼠腋下,建立人胃癌皮下种植动物模型,继而建立人胃癌肿瘤皮下传代动物模型;将皮下传代模型肿瘤组织原位移植高度免疫缺陷小鼠胃部,构建人胃癌原位移植转移动物模型;利用活体成像系统对以上模型进行监测评价。 结果获得稳定表达绿色荧光蛋白和萤火虫荧光素酶的BGC-823-eGFP-luc2细胞株;成功构建皮下种植和传代动物模型;成功构建人胃癌原位移植转移动物模型。 结论慢病毒感染方法适用于人胃癌BGC-823-eGFP- luc2稳转株的建立;利用BGC-823-eGFP-luc2细胞株可以快速构建人胃癌皮下种植动物模型和原位移植转移动物模型,利用活体成像系统可以直观、非侵入的有效监测胃癌远端转移灶的发生、发展情况。     

Articular cartilage repair using tissue engineering technique--novel approach with minimally invasive procedure

Articular cartilage has very limited potential to spontaneously heal, because it lacks vessels and is isolated from systemic regulation. Although there have been many attempts to treat articular cartilage defects, such as drilling, microfracture techniques, soft tissue grafts or osteochondral grafts, no treatment has managed to repair the defects with long-lasting hyaline cartilage. Recently, a regenerative medicine using a tissue engineering technique for cartilage repair has been given much attention in the orthopedic field. In 1994, Brittberg et al. introduced a new cell technology in which chondrocytes expanded in monolayer culture were transplanted into the cartilage defect of the knee. As a second generation of chondrocyte transplantation, since 1996 we have been performing transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints. This signifies a concept shift from cell transplantation to tissue transplantation made ex vivo using tissue engineering techniques. We have reported good clinical results with this surgical treatment. However, extensive basic research is vital to achieve better clinical results with this tissue engineering technique. This article describes our recent research using a minimally invasive tissue engineering technique to promote cartilage regeneration.     

Osteoarthritis year 2010 in review: imaging

Osteoarthritis (OA) is no longer viewed as a passive, degenerative disorder, but rather an active disease process driven primarily by mechanical factors. OA should also be conceptualized as a disease of a whole joint organ, and therefore imaging of OA requires techniques which enable us to visualize the whole joint organ. Although clinical decision making based on imaging findings remains controversial the importance of imaging-derived data in OA research cannot be overemphasized. Since mid-2009, numerous publications reporting on imaging-oriented studies on OA have been reported. These include magnetic resonance (MR) imaging of numerous features of the whole joint such as synovitis, subchondral bone, meniscus, cartilage and cyst-like lesions. Active research is also ongoing using conventional radiography with a focus on measurements of joint space width and alignment of the knee joint. Ultrasound is emerging as a useful imaging technique, particularly in the field of hand OA research. As the importance of imaging-derived data increases, all potential authors are advised that they should seek opinions from expert musculoskeletal radiologist to ascertain the application of correct imaging techniques, especially the MR pulse sequences and image interpretation. The peer-review process of OA imaging in any journal, therefore, should involve musculoskeletal radiologists experienced in OA research to ensure the publication of papers with scientifically sound contents.     

Radiological evaluation of breasts reconstructed with lipomodeling

We have been using the lipomodeling technique since 1999 in order to improve the form and the volume of the breasts reconstructed with latissimus dorsi flap. During several recent years this technique has known a considerable development and its results are quite attractive. At the same time, the breast imaging remains to be a fundamental mean of cancer control. The knowledge of radiological manifestations of the implanted fat tissue is very important. The aim of this study was to evaluate the imaging aspects of lipomodeling in breasts reconstructed with latissimus dorsi flap, using three types of explorations: ultrasound, radiological mammography and MRI. A total of 30 patients were included into the study with average age of 51 years. All of these patients underwent a breast reconstruction using a latissimus dorsi flap with lipomodeling, which average volume reached 165 cc. Four of the patient had a bilateral breast reconstruction. Three modes of imaging (ultrasound, mammography and MRI) were performed a year after the surgery. In 20 patients the imaging findings proved to be normal (in total 34 breasts). An image of fat tissue was revealed in 14 cases and some benign microcalcifications were found in 4 patients. A suspicious tissular image was discovered in one patient, who later underwent a biopsy. The result was a benign giant cell granuloma. In conclusion, the lipomodeling technique does not affect the postoperative follow-up of the patients with breast cancer and an imaging controlled biopsy is possible in case of any doubt. At present, some additional studies are underway evaluating the fat tissue transplants in normal breasts, in order to widen its indications in the plastic breast surgery.     

Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo

Bone is a highly dynamic organ in which several cell types function cooperatively. Among these, osteocytes have recently emerged as an important regulator of bone homeostasis, although their mechanism of regulation is unclear. Here, intravital bone imaging by two-photon excitation microscopy allowed us to directly visualize ‘osteocytic osteolysis’, or resorption of bone in the lacuno-canalicular system. Osteocyte lacunae and the canalicular network in the cortex of murine tibiae were imaged by in vivo calcein staining, and local acidification in these structures was monitored using a topically applied pH sensor. We also demonstrated that sciatic neurectomy causes significant acidification around osteocytic lacunae and enlargement of lacuno-canalicular areas. These results provide strong evidence for osteocytic osteolysis, and demonstrate that two-photon intravital microscopy is useful for analysis of this phenomenon.     

Induction of tumor stem cell differentiation—novel strategy to overcome therapy resistance in gastric cancer

Purpose

Metastases are a frequent finding in gastric cancer and are associated with poor prognosis. A recently discovered link between metabolic changes, differentiation, and therapy resistance due to tumor stem cells could depict a novel approach in cancer research and therapy. Phosphoglycerate kinase 1 (PGK1) is a metabolic enzyme and is known to be involved in enabling gastric cancer cells to be invasive and to disseminate. In this study, we investigated if PGK1 is a promising candidate in inducing stem cell differentiation in gastric cancer.

Materials and methods

MKN45 gastric cancer cells were used due to their known cancer stem cell population, which is defined by the surface marker CD44. MKN45 cells were separated between CD44+ and CD44? cells and, in equal parts, incubated with shRNA anti-PGK1 using fluorescence-activated cell sorting (FACS) analysis; they were then injected into nude mice to evaluate their tumor growth behavior in vivo. Further, the invasive potential of gastric cancer cells was evaluated in vitro using the xCelligence analyzing system.

Results

CD44+ gastric cancer cells treated with and without shRNA anti-PGK1 were capable to cause tumor growth in vivo, whereas tumor growth in CD44+ cells treated with shRNA anti-PGK1 was considerably smaller in comparison with that in CD44+ cells without treatment. CD44? cells did not show any noticeable tumor growth in vivo. By targeting PGK1, the invasive potential of gastric cancer cells was impressively reduced in vitro. In all our cells, which were targeted with shRNA anti-PGK1, we did not find any change that is in accordance with the phenotype of the cells using FACS analysis.

Conclusions

Our findings suggest that targeting the key metabolic enzyme PGK1 in gastric cancer cells may open a new chapter in cancer treatment, which is well worth for further exploration in combination with recent chemotherapy, and might be a promising possibility to overcome therapy resistance in gastric cancer.     

Immunohistochemistry of prostatic acid phosphatase

The human prostatic acid phosphatase is a specific marker for the prostatic epithelial cells. By using an immunoperoxidase staining method for this enzyme, it is possible both to identify the prostatic epithelial cells and to recognize the prostatic origin of metastatic lesions of prostate cancer. Of the tissues containing prostatic epithelial cells from 120 patients, positive staining reaction was detected in 114 (95%), and negative in 6. In nonprostatic tissues from 242 patients, weak but positive staining reaction was detected in 8 (3.3%), including tissues from one renal cell carcinoma and 7 breast carcinomas. Of 27 patients in whom tumor tissues were tested at a time when tumor origin was unknown, the staining reaction was positive in 14 patients later found to have prostate cancer. It was negative in 6 patients with nonprostatic carcinoma and 7 patients with carcinoma of unknown primary. Although this immunohistochemical technique for prostatic acid phosphatase appears promising in diagnosing metastatic prostate cancer, its clinical significance and limitations remain unclear, and there are considerable technical problems yet to be solved. These problems are best approached by joint collaborative efforts of the various investigators interested in prostate cancer.     

Contact laser-assisted neuroendoscopy can be performed safely by using pretreated 'black' fibre tips: experimental data

BACKGROUND AND OBJECTIVE: Laser-assisted endoscopic neurosurgery by using conventional fibres requires the use of high-power laser light. Because this is potentially hazardous, we developed a pretreated fibre tip and evaluated tissue effects in vitro and in vivo. STUDY DESIGN/MATERIALS AND METHODS: By applying a highly absorbing coating to the front of the ball tip, almost all laser light is transformed into thermal energy, instantly producing ablative temperatures at the tip itself. The temperature distribution was examined by using an in vitro thermal imaging technique. The in vivo effect on rabbit cerebral tissue was examined macroscopically and histologically. RESULTS: By using a conventional fibre tip, ablation was not observed, despite the use of high energy and power (20 W for 10 seconds), whereas histology and thermal imaging demonstrated deleterious effects deeply into the cerebral tissue. By using the coated fibre tip, ablation was observed at low energy and power (1 W for 1 second) with thermal effects restricted to superficial structures. CONCLUSIONS: We show that laser-assisted neuroendoscopy can only be considered to be safe when pretreated "black" fibre tips are used, as laser light damages deep structures.     

Video imaging to assess neuromuscular blockade at the larynx

We describe video imaging as a technique for assessing neuromuscular blockade at the larynx. We sought to determine the stability and reproducibility of this technique and to compare the effect of succinylcholine at the adductor pollicis and the larynx. Ten patients were studied. Anesthesia was induced and maintained with propofol. The recurrent laryngeal nerve was stimulated superficially and movements of the vocal cords were recorded on videotape by using a fiberoptic bronchoscope passed via a laryngeal mask airway. Neuromuscular function was recorded at the adductor pollicis by using a mechanomyograph. Twenty images of the vocal cords were examined repeatedly by one investigator and by ten independent observers. The mean difference between the two sets of observations was 0.86 degrees with a correlation coefficient (r) of 0.997. For 3 min before the administration of relaxant the coefficient of variation in the cord movement during supramaximal stimulation ranged from 1%-4% (median 2.7%). After the administration of succinylcholine 1 mg. kg(-1) the times to loss of T1 at the larynx and hand were 63 +/- 15 s and 63 +/- 12 s respectively. Times to 25% recovery were 215 +/- 36 s at the larynx and 436 +/- 74 s at the hand and times to 75% recovery were 285 +/- 55 s and 525 +/- 85 s respectively. These results indicate that video imaging may be a useful research technique for estimating neuromuscular blockade at the larynx and that the time to onset of succinylcholine at the larynx is similar to that at the hand, whereas the duration of blockade is significantly shorter at the larynx. Implications: Assessment of neuromuscular blockade at the larynx is possible by using a video imaging technique. By using this technique, the time to onset of neuromuscular blockade at the larynx is similar to that at the hand after the administration of succinylcholine; this finding is different from previously published data obtained by using a cuff pressure measurement technique.     

High-resolution optical coherence tomography-guided laser ablation of surgical tissue

BACKGROUND. Optical coherence tomography (OCT) is a compact high-speed imaging technology which uses infrared light to acquire cross-sectional images of tissue on the micrometer scale. Because OCT images are based on the optical backscattering properties of tissue, changes in tissue optical properties due to surgical laser ablation should be detectable using this technique. In this work, we examine the feasibility of using real-time OCT imaging to guide the placement and observe the dynamics of surgical laser ablation in a variety of tissue types. MATERIALS AND METHODS. More than 65 sites on five ex vivo rat organ tissue types were imaged at eight frames per second before, during, and after laser ablation. Ablation was performed with a coincident continuous wave argon laser operating at 514-nm wavelength and varying exposure powers and durations. Following imaging, tissue registration was achieved using microinjections of dye followed by routine histologic processing to confirm the morphology of the ablation site. RESULTS. High-speed OCT imaging at eight frames per second permitted rapid tissue orientation and guided ablation in numerous organ specimens. Acquisition rates were fast enough to capture dynamic changes in optical backscatter which corresponded to thermal tissue damage during laser ablation. CONCLUSIONS. The ability of high-resolution high-speed OCT to guide laser ablation and image the dynamic changes suggests a role in image-guided surgical procedures, such as the ablation of neoplasms. Future in vivo studies are necessary to demonstrate performance intraoperatively.     

Diagnosis of Nonmelanoma Skin Cancer/Keratinocyte Carcinoma: A Review of Diagnostic Accuracy of Nonmelanoma Skin Cancer Diagnostic Tests and Technologies

BACKGROUND: Nonmelanoma skin cancer (NMSC) is the most prevalent cancer in the light-skinned population. Noninvasive treatment is increasingly used for NMSC patients with superficial lesions, making the development of noninvasive diagnostic technologies highly relevant. OBJECTIVE: The scope of this review is to present data on the current state-of-the-art diagnostic methods for keratinocyte carcinoma: basal cell carcinoma, squamous cell carcinoma, and actinic keratosis. METHODS AND MATERIALS: MEDLINE, BIOSIS, and EMBASE searches on NMSC and physical and clinical examination, biopsy, molecular marker, ultrasonography, Doppler, optical coherence tomography, dermoscopy, spectroscopy, fluorescence imaging, confocal microscopy, positron emission tomography, computed tomography, magnetic resonance imaging, terahertz imaging, electrical impedance and sensitivity, specificity, and diagnostic accuracy. RESULTS: State-of-the-art diagnostic research has been limited in this field, but encouraging results from the reviewed diagnostic trials have suggested a high diagnostic accuracy for many of the technologies. Most of the studies, however, were pilot or small studies and the results would need to be validated in larger trials. CONCLUSIONS: Some of these new imaging technologies have the capability of providing new, three-dimensional in vivo, in situ understanding of NMSC development over time. Some of the new technologies described here have the potential to make it from the bench to the clinic.     

Diffusion weighted MRI: overview and implications for rectal cancer management

Diffusion weighted imaging (DWI) is an MRI technique that quantifies the movement of water molecules at a cellular level. As the diffusion properties of water vary in areas of necrosis, high cellularity, inflammation and fibrosis, this technique is inherently sensitive to different pathologies. Having become a well‐established adjunct to standard sequences during neurological MRI, technological advances have enabled extrapolation to abdominopelvic imaging, including staging of rectal cancer. Scan acquisitions can be performed rapidly using widely available equipment and consequently there has been rapid dissemination into routine practice. However, while DWI shows promise for detecting, staging and monitoring rectal cancer response to therapy, the evidence base remains scant with no current consensus for technical protocols, interpretation or integration into rectal cancer management. Moreover, those studies available to date have a small sample size and few observers, and their results may not be generalizable to daily practice. This article outlines the physical principles of DWI, reviews the literature and suggests avenues for future research into this important technical development.     

Diagnostic imaging in the preoperative management of lung cancer

Surgical resection is the accepted standard of care for patients with non-small cell lung cancer (NSCLC). Several imaging modalities play central roles in the detection and staging of the disease. The aim of this review is to evaluate the utility of computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and PET/CT for NSCLC staging. Radiographic staging refers to the use of CT as a non-invasive diagnostic technique. However, while the vast majority of patients undergo only CT, CT is a notoriously inaccurate means of tumor and nodal staging in many situations. PET/CT clearly improves the staging, particularly nodal staging, compared to CT or PET alone. In addition, as a result of the increased soft-tissue contrast, MRI is superior to CT for distinguishing between tissue characteristics. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), which is a minimally invasive technique, also has pathological diagnostic potential. Extensive research and the resultant improvements in the understanding of genetics, histology, molecular biology and oncology are transforming our understanding of lung cancer, and it is clear that imaging modalities such as CT, MRI, PET and PET/CT will have an important role in its preoperative management. However, thoracic surgeons should also be aware of the limitations of these techniques.