Article 79

Molecular Imaging of Pancreatic Islet Transplantation

Affiliations

Key words ▶ diabetes mellitus ● ▶ molecular imaging ● ▶ pancreatic islet ● transplantation ▶ contrast agent ● ▶ islet labeling ●

received 15.05.2013 first decision 24.10.2013 accepted 27.11.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1363232 Exp Clin Endocrinol Diabetes 2014; 122: 79–86 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence Prof. B. Song Radiology West China Hospital Guoxue Road No. 37 Chengdu Sichuan Chengdu 610041 China Tel.: + 86/28/8542 3680 Fax: + 86/28/8542 3503 [email protected]

Y. Liu1, B. Song1, X. Ran2, Q. Jiang3, J. Hu4, S. M. Vance Chiang5 1

West China Hospital, Radiology, Chengdu, China West China Hospital, Endocrinology, Chengdu, China 3 Henry Ford Health System, Neurology, Detroit, United States 4 Wayne State University, Detroit, United States 5 West China College of Medicine, Sichuan University, Chengdu, China 2

Abstract

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Islet replacement therapy, pancreatic islet transplantation, is considered as a potential option for curing T1DM. However, the significant loss of implanted islets after islet transplantation prevents it from becoming a mainstream treatment modality. Due to the lack of reliable noninvasive real-time imaging techniques to track the survival of the islets, it is impossible to discover the specific causes for the loss of implanted islets, not to mention taking interventions in the early stage. Current achievements in molecular imaging has provided with several promising techniques, including optical imaging, PET and MRI, for noninvasive visualization, quantification and functional evaluation of transplanted islets in experimental conditions. Optical imaging seems to be the most convenient and costefficient modality, but the limited penetration distance hinders its application in large animal

Introduction

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Diabetes is a chronic disease with incidence rising worldwide. The diabetic population is expected to rise to 552 million by 2 030, up from 366 million in 2011 [1]. A report from the US Centers for Disease Control and Prevention (CDC) recently estimated that a 10-year-old boy or girl diagnosed with diabetes in the year 2000 would lose, on average, 18.7 and 19.0 life-years, respectively, compared with their non-diabetic peers [2]. There are 2 types of diabetes: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). In T1DM, pancreatic β-cell destruction leads to absolute insulin deficiency, which consequently results in hyperglycemia. Although T1DM accounts for a very small proportion of all patients with diabetes, it remains a chronic incurable disorder, with early onset among children and adolescents and substantial risk for

and human studies. PET combined with targetspecific tracers is characterized by high specificity and sensitivity for detection of islet grafts, but observation time is rather short (i. e., several hours). MRI stands out for its long-term visualization of transplanted islet grafts with the aid of contrast agents. However, quantification of islets remains a problem to be solved. A novel technique, microencapsulation, provides a new perspective in multimodal imaging by optimizing the strengths of several modalities together. Although the application of molecular imaging in clinical settings is still limited, significant success and valuable information is achieved in the basic and clinical trials. However, islet transplantation still remains an experimental procedure, with ongoing researches focusing on islets availability, appropriate sites for implantation, new methods using biomaterials (e. g. microencapsulation), immune modulation and more.

long-term morbidity and mortality. The longterm morbidity and mortality of T1DM is mainly associated with diabetic-related complications, which include microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (cardiovascular, cerebrovascular, and peripheral vascular) diseases. Currently all patients with T1DM require insulin either by injection or by pump to control blood glucose [3]. As blood glucose can be affected by many factors including food and drug intake, stress and activity, the control of blood glucose in T1DM is more difficult than T2DM because of the former’s absolute deficiency in innate insulin, which insulin therapy may lead to frequent occurrence of hypoglycemia [4–7]. A new treatment approach, pancreatic islet transplantation, has been introduced to help patients restore glucose homeostasis by resuming the capability of insulin secretion on their own and providing cli-

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nicians and T1DM patients with a potential option for curing insulin-dependent diabetes and preventing diabetes-related complications. The first clinical attempt at islet transplantation was conducted by Watson and Harsant, who transplanted 3 pieces of a sheep’s pancreas beneath the skin of a 15-year-old boy in 1894 [8]. Although the blood glucose level improved, the procedure failed and the patient died 3 days later. Modified and improved for more than a century, the outcome is still far from satisfactory. In 1999, a new protocol, called Edmonton protocol, with reproducible success in terms of insulin independence, gave renewed hope for curing T1DM [9]. By enhancing immunosuppression regimen compared to previous protocols and improving islet delivery strategy, namely, intraportal infusion of freshly isolated islets, followed by a second or likely third infusion of additional islets, 80 % of the patients receiving islet transplantation maintained insulin independence after 1 year. By 2003, a total of 282 sites are involved with islet transplantation. However, embolization via the portal vein into the liver is the only method recommended and used in human trials. From 1999 to 2008, almost 400 patients received allogeneic islet transplants. However, a more recent international trial using the Edmonton protocol indicated that only 44 % of people who received pancreatic islet transplantation remained insulin-independent for more than one year and the reversal percentage reached as high as 80 % after a 5-year clinical follow-up [10]. The reversal of insulin independence is believed to correlate with the loss of islets, which is expected to be associated with various factors. Short-term losses are mainly associated with transplant procedure and the microenvironment of the transplanted site [11], while long-term losses are primarily linked to auto- or alloimmune destruction, immunosuppressive toxicity and progressive disruption of insulin secretion [12]. As there remains a lack of effective noninvasive guiding and tracking technology to monitor the fate of the transplant, assessing its functional status, viability and ultimately, graft outcome after transplantation, early prediction and prevention of islet loss are still not possible. Conventional approaches to evaluation of islet survival and function can be classified as indirect or direct. Indirect means are mostly indicators of glucose metabolism, such as fasting and simulated glucose levels, oral glucose tolerance tests, hemoglobin A1c (HbA1c), C-peptide, and insulin levels [13]. They are easy to perform and can be done repeatedly, but provide little information about the status of one or more specific islets. In addition, it isn’t until a certain number of islets have been destroyed that significant change occurs, making it impossible for early intervention. Liver biopsy is the only direct means of observing the number and appearance of the islet cell directly through a microscope; however, it is time-consuming due to staining of the specimen, so that it can’t be used for real-time monitoring. Also, it is an invasive method with a high risk of severe complications, which makes it impossible for repeated long-term monitoring and clinical follow-up. Moreover, there is also a chance for false negative results due to the heterogenous distribution of implanted islets in the transplanted site [14]. Molecular imaging permits real-time noninvasive imaging of islet transplantation [15], either by optical imaging, positron emission tomography (PET) or magnetic resonance imaging (MRI). Also, combining with certain probes, these modalities aim at guiding the transplantation process, monitoring and quantifying islet graft survival in vivo, and correlating the survival and function of transplanted islets with its anatomical location and route of delivery, which are crucial both for improv-

ing islet transplantation and for monitoring complications posttransplant. Several researchers have provided detailed information on various aspects in this area, including imaging of both endogenous and transplanted islets/beta-cells [13,16–18]. Some articles center on strategies for beta-cell imaging in the context of their clinical relevance [13], the various imaging strategies for assessing islet delivery, outcome of the grafts and immunorejection, or the use of combined imaging and therapeutic interventions [16]. While others emphasized the major achievements of in vivo imaging of islet transplantation [17], obstacles and challenges [19]. Here we will specifically focus on imaging strategies combined with labeling techniques for noninvasive in vivo visualization of transplanted islets from the perspective of molecular imaging, comparing their basic characteristics and applications from basic science to the clinical setting. In order to evaluate the strategy with the greatest potential for clinical application, basic imaging techniques and a novel technology of multimodal imaging, microencapsulation, will be discussed here.

Optical Imaging

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Optical imaging has been widely applied in basic science research for visualization of physiological and pathological processes at the cellular and molecular level in vivo. It is one of the earliest molecular imaging modalities to be applied in islet transplantation and has been widely applied in small animal models for analysis of molecular and genetic expression. Optical imaging has been applied to investigate immune rejection [20], encapsulation technology [21], liver infarction [22] and islet apoptosis [23]. Optical imaging is characterized by its high sensitivity and specificity. It can be performed easily and quickly and costs much less than PET or MRI. Optical imaging mainly includes bioluminescence imaging (BLI) and fluorescence imaging (FI). While BLI utilizes the specific reaction between luciferin and luciferase to release light, fluorescence imaging requires the use of different fluorochromes and excitation light to generate a light signal. Because of their different mechanisms, BLI has a higher specificity than fluorescence imaging, which helps the quantification of the islets [24–27].

Bioluminescence imaging (BLI) BLI requires a luciferase gene to label cells ex vivo. When the labeled cells are transplanted into the recipient, luciferase transforms its substrate, luciferin, into oxyluciferin while at the same time releasing photons which can be detected by sensitive devices such as a charge-coupled device (CCD) camera, enabling detection of cellular and genetic activity. BLI has many advantages. Firstly, it allows long-term observation. Lu et al. transduced isolated rodent and human islets with modified adenovirus or lentivirus carriers that expressed a luciferase gene and transplanted them separately into suprarenal capsules [24]. The signal from the islet engineered with the lentivirus lasted 140 days, whereas adenovirus-directed luciferase expression rapidly attenuated. In other studies, a stable signal could be monitored for more than 1 year, with the longest signal lasting up to 18 months [25, 27]. At the end of these studies, histological analysis confirmed that viral transduction, luciferase expression, and repeated imaging had no deleterious effects on islet function during the observation period [24]. A second advantage of BLI is high sensitivity. Chen et al. detected BLI signals in as few as 10 islets implanted in the renal subcapsular

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Fluorescence imaging (FI) FI requires different fluorochromes (fluorescent proteins, dyes et al.) and an excitation light to illuminate the subject and generate the light signal. This light signal can also be detected by a chargecoupled device (CCD) camera, unlike BLI which exploits the specific reaction between luciferase and luciferin. Researchers have generated a transgenic mouse strain expressing green fluorescent protein under control of the mouse insulin I promoter to visualize histological and pathological changes in intraportally transplanted islets and surrounding hepatic tissue [22]. The transgenic mouse can also be tagged with red or cyan fluorescent protein [28]. With the help of reflected light confocal imaging and freely available software (i. e., MetaMorph or Voxx software), it is possible to acquire information in 3-dimensions, as opposed to conventional immunohistochemistry which is time-consuming and 2-dimensional. Compared to BLI, FI is easily performed and does not require injecting a substrate. Moreover, it is possible to detect regions of islet apoptosis both in vitro and ex vivo with intravenous administration of a near-infrared probe (i. e., annexin V Cy5.5) [23]. However, as tissues in the body can generate light, quantification of targeted cells may be difficult. Thus FI is much less specific than BLI and its application in islet transplantation is rather limited. Also, as with BLI, FI suffers from limited penetration, which is even less than BLI. So the tissue overlying the fluorescent islets must be removed in order to be monitored. Although optical imaging seems difficult to transfer from basic science research into clinical application, it has offered a lot of information on early status and changes in the implanted islets. Optical imaging is still being employed to study islets in the pancreas in vivo in small animals and has great value in evaluating histological and pathological changes. As for clinical application, radionuclide and magnetic radial imaging are preferred.

PET

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PET is widely applicable to noninvasively monitor cell metabolism and function, and along with MRI, they have been successfully transferred from basic experiments to clinical trials in the field of islet transplantation [29, 30]. PET has high sensitivity (i. e., 10 − 11-10 − 12 mol/l), 6–8 times higher than that of MRI [17, 31]. And by using target-specific tracers, the specificity of PET imaging can be significantly improved, permitting longitudinal quantification of targeted cells. The most frequently used radiotracer in pancreatic islet transplantation is 18F-fluorodexyglucose ([18F]–FDG) which is readily available and widely used in the clinic. Before transplantation, the isolated islet cells need to be labeled with the radiotracer ex vivo. Once entering the islet cell, [18F]-FDG is phosphorylated into [18F]-FDG-6P [13], which can neither be metabolized nor transferred to the outside of the cell. So [18F]-FDG-6P is trapped inside the islet cell. When the cell dies, the released [18F]-FDG6P is not taken up by other cells but becomes distributed throughout the body without specific accumulation in other organs, which increases accuracy of imaging the transplanted islets [13, 14, 29]. Tracking cells labeled with a radioactive tracer has several advantages. It offers high sensitivity and specificity, and reportedly [18F] -FDG allows detection and quantification of signals as small as 3 mm in size [32]. Moreover, it facilitates quantification of the islet graft, as the signal detected by PET/SPECT correlates directly to the quantity of the transplanted islets. With the use of radiotracers, researchers are able to explore various aspects of transplanted islets. In a study performed by Eriksson, they found the distribution of the transplanted islet cells in the liver to be heterogeneous in a large animal model (i. e., pig), with no accumulation in other organs [14]. As with optical imaging, the magnitude (radioactivity) of transplanted islets decreased by almost 50 % within just a few minutes after implantation using syngeneic transplant. Other researchers reported similar findings [29, 30]. However, the short half-life of radioactive tracers limits further investigation. The half-life of 18 F is 109.8 min and the reported detection time of [18F]-FDG was limited to 6 h [32]. This makes it impossible for longitudinal observation of the graft and analysis of possible causes of rejection, including autoimmune reaction, radioactivity and compromised blood flow of the portal vein. But little is known about the status of and damage to the islets. Other isotopes with a longer half-life have been studied; for example, 64Cu showed promise in detecting islet graft and subcutaneous insulinoma in mice [33]. However, the imaging time was still limited to 24–36 h, which is far too short for long-time monitoring. Fortunately, transgenic technology offers a way of prolonging observation time by using a reporter gene to modify the cell of interest. Herpes simplex virus 1thymidine kinase (HSV-1tk) is a commonly used reporter gene that can be carried by an adenovirus or lentivirus to transfect islet cells ex vivo, when the modified islets are engrafted in the body. After engraftment, the radiotracer [124I]-5-iodo-1-(2-deoxy-2-fluoro-D-arabino-furanosyl) uracil is administered. The radiotracer is then phosphorylated by thymidine kinase once inside viable islet cells, and the amount of tracer retained by the islets in vivo can be measured by PET [13]. Using reporter genes, the observation time is longer than with radioactive tracers. As HSV1-sr39Tk, a mutant form of HSV-1TK, can phosphorylate [18F]-FHBG more effectively [34], Lu et al.

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space, intrahepatic, intraabdominal, and subcutaneous locations [25]. Also, because of the specific reaction between luciferase and luciferin, and the low background in the body, transplanted islets can easily be observed and the magnitude of the signal analyzed. Several studies have found a linear correlation between the number of transplanted islets and the magnitude of the signal [24–27], which confirmed direct quantification of the islet grafts. However, after normoglycemia and stable luminescence intensity were achieved, bioluminescent intensity in the allograft progressively decreased, followed by recurrence of hyperglycemia which indicated loss of the graft [25]. Histological analysis confirmed the rejection. BLI has been applied in different aspects of islet transplantation. Chen et al. investigated the effect of immune rejection on functional islet mass and correlated it with metabolic abnormalities between isograft and allograft [20]. While Roth et al. applied BLI to evaluate the biocompatibility of alginate-based capsules in islet transplantation [21]. Although BLI seems quite promising due to its long observation time, high specificity and sensitivity, and precise quantification compared to other imaging methods, researchers found that serum glucose level, surgical process, transplant site and attenuation of photons could have a significant impact on the detected signal, making analysis complicated [21]. More importantly, as photons can be absorbed by organic tissue, the light can only penetrate several centimeters of the tissue, which hinders its application in large animals and humans.

used HSV1-sr39Tk, engineered with a lentivirus, to combine with a PET radiotracer, 9-(4-[18F]-fluoro-3-hydromethylbutyl) guanine ([18F]-FHBG). The islet cells were monitored by microPET. Their results showed that the transfected islet grafts transplanted in the axillary cavity of NOD-scid (non-obese diabetic/ severe combined immunodeficient) mice could be repeatedly detected by microPET for at least 90 days compared with only 40 days with the adenovirus [35, 36]. They reported that there was a significant and almost proportional relationship between the intensity of the signal and the number of transplanted islets [34]. Another study confirmed the direct proportionality using a therapeutic gene, interleukin-10, during transplantation into the liver [36]. However, signal intensity decreased by 50 % during the first few weeks – indicating the death of the islets – and thereafter remained stable after about 30 days. While these researchers assumed this was due to the less supportive environment of the axillary cavity [34], the explanation is far more complicated. After they compared the early decrease in the signal with nonfasting blood glucose levels, intraperitoneal glucose tolerance tests, plasma insulin levels and immunohistochemistry (IHC), they found that the signal loss was caused by rejection of the graft. Although reporter genes offer a much longer observation time than radiotracers and histological analysis showed that the genes have no effect on cell viability and function, their longterm influence on the engineered cells is still not clear. Some researchers wonder if the reporter gene has the potential to manipulate the genes of the cells [13], which would limit its further application in humans. Also, exposure to ionizing radiations from PET has to be considered while imaging in the clinic.

Magnetic Resonance Imaging (MRI)

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Compared with other tomographic imaging modalities, MRI delivers the best spatial resolution, soft-tissue contrast and unlimited penetration. It has near-microscopic resolution and can offer information regarding anatomical and physiological parameters at the same time. Also, due to its use of non-ionizing radiations, it can be used repeatedly to guide real-time islet transplantation and visualize islet grafts in vivo. However, MR imaging is less sensitive in detecting molecular probes than PET and optical imaging [31]. Although various MR imaging strategies and the increasing of the field strength of the MR scanner can improve the sensitivity to some extent, the use of contrast agents can greatly enhance the detection limit even under a relatively low magnetic strength MR scanner, which is critical for clinical use. The most popular contrast agents for MRI include superparamagnetic iron-oxide (SPIO) nanoparticles, fluoride-19 and gadolinium (Gd). As T2 contrast agents, SPIOs are shown as hypointensive areas. While fluoride-19 and Gd, as T1 contrast agents, are shown as hyperintensive areas.

Superparamagnetic iron-oxide (SPIO) nanoparticles SPIOs are the most popular T2 and T2*-weighted (negative) contrast agents for islet graft imaging. Their main structure typically consists of an iron oxide core covered with a dextran coat. The FDA-approved commercially available SPIO is called ‘Feridex’, which is often used in the clinic for liver imaging. The coat can be modified to develop various types of SPIOs for MR imaging with different ex vivo label time and efficiency, such as the carboxydextran-coated SPIO nanoparticle ferucarbotran (com-

mercially called ‘Resovist’, a new clinically-approved MRI contrast agent) [37], immunomagnetic antibody-coated iron beads [38], chitosan-coated SPIOs [39], heparinized SPIOs [40], ferrimagnetic iron oxide nanocubes (FIONs) [41], polyvinylpyrrolidone-coated SPIOs [42], et al. The most prominent feature of SPIO-labeled MR imaging in the field of transplanted islet imaging is that it enables long-term observation of transplanted pancreatic islets. Jirak et al. first used Resovist in a rat model of intrahepatic islet transplantation; the labeled islets were seen as hypointense foci in T2-weighted images at 4.7T MR [43] and the signal lasted for 22 weeks posttransplantation. The longest reported signal intensity of SPIOlabeled islets were observed by Evgenov et al., which persisted for 188 days post-engraftment [44]. The diabetic mice became normoglycemic until the end of the observation period. Similar results were obtained by the same group in islet transplantation to the liver, and by other groups as well [45]. Although a previous study suggested that labeling pancreatic islet cells with SPIOs led to decreased insulin secretion compared with unlabeled cells [43], thus far SPIOs have been shown to have no negative effect on the biology and function of labeled islet cells. Kim et al. showed that iron particles had no harmful effect on gene expression of the islets using reverse transcriptase polymerase chain reaction [37]. Relative loss of the islet signal was observed in allogenic SPIOlabeled islet transplantation in non-diabetic animals, the same as optical and radionuclide imaging. In Evgenov’s study, the loss of islets in immunocompetent mice in the early stage after transplantation was 20 % higher than in immunocompromised animals [46], suggesting a significant role of immune rejection. Other factors might include ischemia and non-alloantigen-specific inflammatory events in the liver after transplantation. More recently researchers observed a similar phenomenon while performing autologous islet grafts in the liver and under the renal capsule in non-human primates [18]. Most of these studies were performed in non-diabetic animals; when the complex pathophysiological changes caused by hyperglycemia are taken into account, the situation was even worse [47, 48]. Loss of transplanted islets is much higher in diabetic models, as consistently high levels of blood glucose may be toxic to the graft. In Kim’s study, MR signal intensity of labeled islets decreased by 80 % over 30 days in a diabetic mouse model [48]. In order to be clinically applicable, low magnetic strength machines need to be implemented to evaluate the graft as well, which means that both spatial resolution and sensitivity of SPIOs will significantly decrease. Tai et al. first imaged SPIOtagged islets at 1.5 T, and as few as 200 syngeneic islets under the renal capsule were successfully detected in healthy rats [49]; nevertheless, the transplanted islets clustered together, forming a so-called “blooming artifact” in a signal void. The signal void was larger than the actual area occupied by the labeled cells, making it difficult to quantify. Another necessity for clinical application is to carry out pancreatic islet transplantation in large animal models to mimic a more complex physiological environment similar to human beings. The first large animal study was performed by in 2007 by Barnett et al. in swine [50]. They encapsulated human islets in immunoprotective magnetocapsules containing Feridex. The contrast agent within the capsule permitted them to track the initial islet – containing magnetocapsule infusion and engraftment into the liver. Although their study did not show the islets directly, it demonstrated for the first time the usefulness of a clinical scanner for

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detection of single encapsulated islets in large animals. Recently Moore et al. reported in vivo imaging of autologous islet grafts transplanted in the liver and under the renal capsule of nonhuman primates using a 1.5 T MR scanner [18]. The presence and distribution of islet grafts beneath the capsule and throughout the hepatic parenchyma were clearly seen under a low-field clinical scanner. Although SPIOs seem quite promising in the application for imaging of the islets, quantification of transplanted islets using SPIOs in both animal and human studies remains a big problem. The area of contrast exceeds the size of the islets themselves, so that a single hypointense spot can represent either a single labeled islet or a cluster of many closely spaced islets [38]. Also, the wide distribution of implanted islets in the liver makes it much more difficult to count. Moreover, SPIOs result in ‘negative contrast’ on MR images: they appear as hypointense spots that can be difficult to distinguish from the heterogeneous hepatic MRI background, especially in the iron over-load background. Kim et al. [51] found that excluding small hypointense spots on MR imaging could improve the association between the number of hypointense spots and the blood glucose level of the recipient. This could potentially be a useful principle in developing an algorithm for estimating islet mass, but it requires further verification. Other methods involve using an algorithm to estimate islet mass instead of counting them manually. In their nonhuman primate study, Moore et al. developed a semi-automated image segmentation algorithm for identification of hypointense voxels representing islet grafts on MR images and quantitative analysis of their relative abundance over time. This can be used to estimate and track relative transplanted islet mass by MRI [18]. Crowe et al. used a 3D radial ultra-short echo time difference technique with a 3T clinical scanner in a rodent model, rendering the labeled cells as positive contrast on the images, suppressing liver signal and small vessels and allowing automatic quantification [52]. Computational analysis offers a new method of automatic quantification which is time-saving and productive and could potentially be applied in the clinical situation as well.

edly up to 65 days for transplanted human islet cells into the kidney of SCID mice using a Gd chelate, GdHPDO3A [45]. While signal intensity decreased after 10 days posttransplantation – similar to SPIOs – it was also found to increase with time during the first week. The researchers assumed this might be attributable to the intracellular redistribution of the islets from the cytoplasm, but it could also be due to release of the dying islets into the intercellular space resulting from the transplant process, immune rejection and other factors as other studies suggested. So the relationship between signal intensity and the remaining islets is difficult to interpret. Another application of Gd is as a blood pool agent. Hathout et al. first employed an FDA-approved Gd complex, gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), which is widely used in the clinic, in a DCE-MRI study to evaluate neovascularization of the transplanted islet graft [55]. The results showed that transplanted islets could be visualized on day 3 without being labeled with an MRI contrast agent. As formation of a new vascular network is crucial for survival of the transplanted islet graft, monitoring the process might provide insight into the viability and function of the graft, and an increase in signal intensity would indicate establishment of a new vascular network. Another similar study was carried out by Chan et al., using the same technique to establish a timeline of vascularization in intraportal islet transplantation [56]. They observed 2 peak signal intensities and compared them with immune stains: the first peak resulted from formation of small vessels in the islets, while the far smaller second peak was confirmed to be emergence of peri-islet vessels. By comparing the histological angiogenesis findings with the signal intensity of DCE-MRI, researchers established a positive correlation between contrast enhancement and increase in new peri-islet microvessels. Although the observation time for the vascularization of the islets was shorter than directly observing them with a contrast agent, it offers a new method for evaluating early survival of islets. Despite this success, there are certain concerns about the side effects of Gd. Its long-term retention in the body might potentially lead to complications such as renal fibrosis in dialysis patients and patients with severe renal failure [57–59].

Fluoride-19 Although SPIOs are the premier MR imaging contrast agents explored in the field of transplanted islet imaging, T1 contrast agents are also being investigated to avoid the pitfalls encountered with T2 contrast agents, such as fluoride-19. As a common T1 contrast agent, the labeled cells present as ‘positive hot spots’ on 19F MR images, in contrast with the hypointense signal presented by T2 contrast agents. Because of the lack of endogenous fluoride in the body, there is no background disturbance in visualization of transplanted labeled islets, making it easy to quantify them under 19F MRI. Thus it affords reliable estimation of the apparent number of islets from image data. Barnett et al. used perfluoropolyether and perfluorooctylbromide to image the implanted islets with 3 modalities: 19F NMR or 19F MRI, ultrasound and CT [53] and found that 19F signal intensity was linear with the number of labeled islets. The same group also combined this technology with microcapsulation for immunoprotection of xenografted islets with multimodal imaging [54].

Gadolinium (Gd) Gadolinium (Gd) is another popular T1 (positive) MR contrast agent which has a shorter observation time than SPIOs, report-

Multimodal Imaging

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As all imaging modalities have pros and cons, microencapsulation technology has opened up a new avenue for multimodal imaging by optimizing their collective strengths. Both islets and contrast agents are contained inside a bio-compatible capsule so that different modalities can be used to monitor transplanted islets. The wall of the capsule consists of a thin alginate membrane which is semi-permeable, allowing smaller molecules (e. g. insulin, metabolites et al.) to pass, while blocking entry of larger molecules (e. g. antibodies, immune cells et al.). The capsule wall does not affect normal islet function and can prevent them from immune rejection without the need for immunosuppressive drugs. While this was its initial purpose, researchers affirmed its versatility in multimodal imaging by either containing contrast agents inside the capsule or incorporating them onto its surface. Various imaging markers have been co-encapsulated with islets, such as SPIOs integrated with near-infrared fluorescent Cy5.5 dye for 1H MRI and optical imaging [44], perfluorocarbons for 19F MRI, ultrasound, and CT [60], and gold nanoparticles coated

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with Gd chelates for 1H MRI, CT and ultrasound [61]. The inside of the capsule can also be modified into a protein microfiber network to better support the embedded islets [62]. Moreover, Kim et al. developed “capsule-in-capsules” containing gold and iron nanoparticles for MRI, CT and ultrasound [63], in which the islets were not only separated from the immune system but also from the nanoparticles to prevent potential toxic effects of the iron. By combining different imaging modalities, their strengths can be maximized. For example, one can use ultrasound to guide the process, while employing MRI or CT to track the distribution and longitudinal outcome of the implanted islets. Also, with the immunoprotective quality of microencapsulation technology, it can not only improve the outcome but avoid the high cost and side effects of immunosuppressive drugs. However, this method does not allow direct visualization of the viable islets, as contrast agents are embedded in the capsule or incorporated onto the capsules instead of directly labeling the islets. The loss of islets won’t be shown on the images; and only when the capsule ruptures a loss of signal can be detected. Moreover, although some researchers have demonstrated the safety of magnetocapsules on islets [53, 64], the potential long-term negative effects of imaging contrast agents preserved in the capsules on islets as well as the surrounding tissue remain unknown. Nevertheless, microencapsulation still remains the most promising technique for islet transplantation imaging and therapeutic intervention in the clinical setting.

Clinical Application

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The first clinical study that we know of using [18F] -FDG as the radiotracer with PET for islet imaging was carried out in 2007 on one patient by Eriksson et al. [30]. In 2009, the same group performed intrahepatic islet transplantation via the portal vein in five diabetic patients using [18F]-FDG-labeled islet cells and performed real-time monitoring and quantification 1 h after transplantation [29]. They reported that the implanted islets were heterogeneous in the liver, and that early loss (i. e., 25 %) of implanted islets was detected just a few minutes after transplantation. HbA1c values of all patients were reduced and the dose of insulin they used was less than before transplantation, indicating viability and normal function of the graft. Fasting C-peptides increased adequately after meal stimulation. However, as visualization time was limited, specific information about the transplanted islets and their fate could not be obtained. The clinical application of MRI with SPIOs in human pancreatic islet transplantation was conducted in 2008 by Toso et al. [65]. The labeled islets were transplanted into 4 patients with T1DM. All patients reached insulin independence, with normal HbA1c after transplantation, and with the longest insulin independence up to 24 months post-transplant. Due to spontaneous high iron content, one patient had diffuse hypointense images on her baseline liver MRI and transplant-related alterations could not be observed. The other 3 out of 4 patients had normal intensity on pretransplant images, and iron-loaded islets could be recognized as hypointense spots within the liver after transplantation. Hyperintense spots could be identified up to 6 months after transplantation. This confirmed the benign application of SPIOs to label islets as well as for long-time observation. However, this study did not find any correlation between the number of

labeled transplanted islets and hypointense spots on MR images, and neither did another human study using SPIOs [66]. In addition, the number of hypointense spots varied significantly over the course of the study, making it impossible to predict graft outcome. Taken together, the application of molecular imaging in clinical trials using MRI and PET is limited, although a considerable number of studies have been carried out. But valuable information was acquired, which needs to be repeated in larger studies with more subjects. Moreover, there are also some obstacles in the area of islet transplantation, such as the lack of islet availability (i. e., either from donors or stem cells), the sideeffects (such as bleeding, mouth ulcers, diarrhea, anaemia, and so forth) both from the procedure and from the use of immunosuppressive drugs. Islet transplantation still remains an experimental procedure, with ongoing research focusing on the islets derived from stem cell technology, appropriate sites for implantation, new methodusing biomaterials (i. e., microencapsulation), immune modulation, improved vascularization, and more.

Conclusions

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Current achievements in molecular imaging modalities and labeling techniques allow noninvasive visualization, quantification, and functional evaluation of transplanted islet grafts. Optical imaging seems to be the most convenient and cost-efficient modality for imaging transplanted islets, and it has been widely applied in molecular and cellular research. However, the limited penetration distance blocks its application in large animal and human studies. PET combined with target-specific tracers is characterized by high specificity and sensitivity for detection of islet grafts, but observation time is rather limited (i. e., several hours). Although reporter genes prolong visualization to several months, the potential of gene manipulation makes it impossible to be applied in human beings. So PET can only be used for short-term observation of islets in basic science and clinical research into islet transplantation. MRI stands out for its longterm visualization of transplanted islet grafts with the aid of SPIOs. It has been applied to experiments in both large and small animals, normal as well as diabetic models, high and low magnetic fields, basic science studies, as well as clinical applications. However, quantification of islets in MRI with SPIOs still remains a problem to be solved. Microencapsulation provides a new perspective of multimodal imaging by optimizing the strengths of several modalities together; but it does not permit direct visualization of viable islets. Some researchers even speculate on the possibility of applying PET/MRI for both acute and long-term engraftment imaging by dual labeling of islets using [18-F]-FDG in combination with paramagnetic nanoparticles. In conclusion, molecular imaging has greatly advanced investigation into the mechanisms and pathophysiological changes related to the condition and outcome of islet grafts, which would benefit not only for to the improving of the transplant procedure, but also to the preventing of the loss of transplanted islets by performing early interventions. Therefore, it represents a bridge connecting basic science to clinical application, making it a perfect candidate for the guidance, monitoring and tracking of islet transplantation.

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Acknowledgement

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None

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