Normal flow velocities and Resistance Index in orbital vessels [15, 31, 32].
\\n\\n
Dr. Pletser’s experience includes 30 years of working with the European Space Agency as a Senior Physicist/Engineer and coordinating their parabolic flight campaigns, and he is the Guinness World Record holder for the most number of aircraft flown (12) in parabolas, personally logging more than 7,300 parabolas.
\\n\\nSeeing the 5,000th book published makes us at the same time proud, happy, humble, and grateful. This is a great opportunity to stop and celebrate what we have done so far, but is also an opportunity to engage even more, grow, and succeed. It wouldn't be possible to get here without the synergy of team members’ hard work and authors and editors who devote time and their expertise into Open Access book publishing with us.
\\n\\nOver these years, we have gone from pioneering the scientific Open Access book publishing field to being the world’s largest Open Access book publisher. Nonetheless, our vision has remained the same: to meet the challenges of making relevant knowledge available to the worldwide community under the Open Access model.
\\n\\nWe are excited about the present, and we look forward to sharing many more successes in the future.
\\n\\nThank you all for being part of the journey. 5,000 times thank you!
\\n\\nNow with 5,000 titles available Open Access, which one will you read next?
\\n\\nRead, share and download for free: https://www.intechopen.com/books
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Preparation of Space Experiments edited by international leading expert Dr. Vladimir Pletser, Director of Space Training Operations at Blue Abyss is the 5,000th Open Access book published by IntechOpen and our milestone publication!
\n\n"This book presents some of the current trends in space microgravity research. The eleven chapters introduce various facets of space research in physical sciences, human physiology and technology developed using the microgravity environment not only to improve our fundamental understanding in these domains but also to adapt this new knowledge for application on earth." says the editor. Listen what else Dr. Pletser has to say...
\n\n\n\nDr. Pletser’s experience includes 30 years of working with the European Space Agency as a Senior Physicist/Engineer and coordinating their parabolic flight campaigns, and he is the Guinness World Record holder for the most number of aircraft flown (12) in parabolas, personally logging more than 7,300 parabolas.
\n\nSeeing the 5,000th book published makes us at the same time proud, happy, humble, and grateful. This is a great opportunity to stop and celebrate what we have done so far, but is also an opportunity to engage even more, grow, and succeed. It wouldn't be possible to get here without the synergy of team members’ hard work and authors and editors who devote time and their expertise into Open Access book publishing with us.
\n\nOver these years, we have gone from pioneering the scientific Open Access book publishing field to being the world’s largest Open Access book publisher. Nonetheless, our vision has remained the same: to meet the challenges of making relevant knowledge available to the worldwide community under the Open Access model.
\n\nWe are excited about the present, and we look forward to sharing many more successes in the future.
\n\nThank you all for being part of the journey. 5,000 times thank you!
\n\nNow with 5,000 titles available Open Access, which one will you read next?
\n\nRead, share and download for free: https://www.intechopen.com/books
\n\n\n\n
\n'}],latestNews:[{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"}]},book:{item:{type:"book",id:"7360",leadTitle:null,fullTitle:"Fillers - Synthesis, Characterization and Industrial Application",title:"Fillers",subtitle:"Synthesis, Characterization and Industrial Application",reviewType:"peer-reviewed",abstract:"Fillers - Synthesis, Characterization and Industrial Application comprises a set of chapters that brings an interdisciplinary perspective to accomplish a more detailed understanding of filler materials for the synthesis and characterization of different industrial applications. 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Neuromuscular disease is a very broad term that encompasses many diseases and aliments that either directly, via intrinsic muscle pathology, or indirectly, via nerve pathology, impair the functioning of the muscles. Neuromuscular diseases affect the muscles and/or their nervous control and lead to problems with movement. Many are genetic; sometimes, an immune system disorder can cause them. As they have no cure, the aim of clinical treatment is to improve symptoms, increase mobility and lengthen life. Some of them affect the anterior horn cell, and are classified as acquired (e.g. poliomyelitis) and hereditary (e.g. spinal muscular atrophy) diseases. SMA is a genetic disease that attacks nerve cells, called motor neurons, in the spinal cord. As a consequence of the lost of the neurons, muscles weakness becomes to be evident, affecting walking, crawling, breathing, swallowing and head and neck control. Neuropathies affect the peripheral nerve and are divided into demyelinating (e.g. leucodystrophies) and axonal (e.g. porphyria) diseases. Charcot-Marie-Tooth (CMT) is the most frequent hereditary form among the neuropathies and it’s characterized by a wide range of symptoms so that CMT-1a is classified as demyelinating and CMT-2 as axonal (Marchesi & Pareyson, 2010). Defects in neuromuscular junctions cause infantile and non-infantile Botulism and Myasthenia Gravis (MG). MG is a antibody-mediated autoimmune disorder of the neuromuscular junction (NMJ) (Drachman, 1994; Meriggioli & Sanders, 2009). In most cases, it is caused by pathogenic autoantibodies directed towards the skeletal muscle acetylcholine receptor (AChR) (Patrick & Lindstrom, 1973) while in others, non-AChR components of the postsynaptic muscle endplate, such as the muscle-specific receptor tyrosine kinase (MUSK), might serve as targets for the autoimmune attack (Hoch et al., 2001). Although the precise origin of the autoimmune response in MG is not known, genetic predisposition and abnormalities of the thymus gland such as hyperplasia and neoplasia could have an important role in the onset of the disease (Berrih et al., 1984; Roxanis et al., 2001).
Several diseases affect muscles: they are classified as acquired (e.g. dermatomyositis and polymyositis) and hereditary (e.g. myotonic disorders and myopaties) forms. Among the myopaties, muscular dystrophies are characterized by the primary wasting of skeletal muscle, caused by mutations in the proteins that form the link between the cytoskeleton and the basal lamina (Cossu & Sampaolesi, 2007). Mutations in the dystrophin gene cause severe form of hereditary muscular diseases; the most common are Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). DMD patients suffer for complete lack of dystrophin that causes progressive degeneration, muscle wasting and death into the second/third decade of life. Beside, BMD patients show a very mild phenotype, often asymptomatic primarily due to the expression of shorter dystrophin mRNA transcripts that maintain the coding reading frame. DMD patients’ muscles show absence of dystrophin and presence of endomysial fibrosis, small fibers rounded and muscle fiber degeneration/regeneration. Untreated, boys with DMD become progressively weak during their childhood and stop ambulation at a mean age of 9 years, later with corticosteroid treatment (12/13 yrs). Proximal weakness affects symmetrically the lower (such as quadriceps and gluteus) before the upper extremities, with progression to the point of wheelchair dependence. Eventually distal lower and then upper limb weakness occurs. Weakness of neck flexors is often present at the beginning, and most patients with DMD have never been able to jump. Wrist and hand muscles are involved later, allowing the patients to keep their autonomy in transfers using a joystick to guide their wheelchair. Musculoskeletal contractures (ankle, knees and hips) and learning difficulties can complicate the clinical expression of the disease. Besides this weakness distribution in the same patient, a deep variability among patients does exist. They could express a mild phenotype, between Becker and Duchenne dystrophy, or a really severe form, with the loss of deambulation at 7-8 years. Confinement to a wheelchair is followed by the development of scoliosis, respiratory failure and cardiomyopathy. In 90% of people death is directly related to chronic respiratory insufficiency (Rideau et al., 1983). The identification and characterization of dystrophin gene led to the development of potential treatments for this disorder (Bertoni, 2008). Even if only corticosteroids were proven to be effective on DMD patient (Hyser and Mendell, 1988), different therapeutic approaches were attempted, as described in detail below (see section 7).
The identification and characterization of the genes whose mutations caused the most common neuromuscular diseases led to the development of potential treatments for those disorders. Gene therapy for neuromuscular disorders embraced several concepts, including replacing and repairing a defective gene or modifying or enhancing cellular performance, using gene that is not directly related to the underlying defect (Shavlakadze et al., 2004). As an example, the finding that DMD pathology was caused by mutations in the dystrophin gene allowed the rising of different therapeutic approaches including growth-modulating agents that increase muscle regeneration and delay muscle fibrosis (Tinsley et al., 1998), powerful antisense oligonucleotides with exon-skipping capacity (Mc Clorey et al., 2006), anti-inflammatory or second-messenger signal-modulating agents that affect immune responses (Biggar et al., 2006), agents designed to suppress stop codon mutations (Hamed, 2006). Viral and non-viral vectors were used to deliver the full-length - or restricted versions - of the dystrophin gene into stem cells; alternatively, specific antisense oligonucleotides were designed to mask the putative splicing sites of exons in the mutated region of the primary RNA transcript whose removal would re-establish a correct reading frame. In parallel, the biology of stem cells and their role in regeneration were the subject of intensive and extensive research in many laboratories around the world because of the promise of stem cells as therapeutic agents to regenerate tissues damaged by disease or injury (Fuchs and Segre, 2000; Weissman, 2000). This research constituted a significant part of the rapidly developing field of regenerative biology and medicine, and the combination of gene and cell therapy arose as one of the most suitable possibility to treat degenerative disorders. Several works were published in which stem cell were genetically modified by ex vivo introduction of corrective genes and then transplanted in donor dystrophic animal models.
Stem cells received much attention because of their potential use in cell-based therapies for human disease such as leukaemia (Owonikoko et al., 2007), Parkinson’s disease (Singh et al., 2007), and neuromuscular disorders (Endo, 2007; Nowak and Davies, 2004). The main advantage of stem cells rather than the other cells of the body is that they can replenish their numbers for long periods through cell division and, they can produce a progeny that can differentiate into multiple cell lineages with specific functions (Bertoni, 2008). The candidate stem cell had to be easy to extract, maintaining the capacity of myogenic conversion when transplanted into the host muscle and also the survival and the subsequent migration from the site of injection to the compromise muscles of the body (Price et al., 2007). With the advent of more sensitive markers, stem cell populations suitable for clinical experiments were found to derive from multiple region of the body at various stage of development. Numerous studies showed that the regenerative capacity of stem cells resided in the environmental microniche and its regulation. This way, it could be important to better elucidate the molecular composition – cytokines, growth factors, cell adhesion molecules and extracellular matrix molecules - and interactions of the different microniches that regulate stem cell development (Stocum, 2001).
Several groups published different works concerning adult stem cells such as muscle-derived stem cells (Qu-Petersen et al., 2002), mesoangioblasts (Cossu and Bianco, 2003), blood- (Gavina et al., 2006) and muscle (Benchaouir et al., 2007)-derived CD133+ stem cells. Although some of them are able to migrate through the vasculature (Benchaouir et al., 2007; Galvez et al., 2006; Gavina et al., 2006) and efforts were done to increase their migratory ability (Lafreniere et al., 2006; Torrente et al., 2003a), poor results were obtained.
Embryonic and adult stem cells differ significantly in regard to their differentiation potential and in vitro expansion capability. While adult stem cells constitute a reservoir for tissue regeneration throughout the adult life, they are tissue-specific and possess limited capacity to be expanded ex vivo. Embryonic Stem (ES) cells are derived from the inner cell mass of blastocyst embryos and, by definition, are capable of unlimited in vitro self-renewal and have the ability to differentiate into any cell type of the body (Darabi et al., 2008b). ES cells, together with recently identified iPS cells, are now broadly and extensively studied for their applications in clinical studies.
Embryonic stem cells are pluripotent cells derived from the early embryo that are characterized by the ability to proliferate over prolonged periods of culture remaining undifferentiated and maintaining a stable karyotype (Amit and Itskovitz-Eldor, 2002; Carpenter et al., 2003; Hoffman and Carpenter, 2005). They are capable of differentiating into cells present in all 3 embryonic germ layers, namely ectoderm, mesoderm, and endoderm, and are characterized by self-renewal, immortality, and pluripotency (Strulovici et al., 2007).
hESCs are derived by microsurgical removal of cells from the inner cell mass of a blastocyst stage embryo (Fig. 1). The ES cells can be also obtained from single blastomeres. This technique creates ES cells from a single blastomere directly removed from the embryo bypassing the ethical issue of embryo destruction (Klimanskaya et al., 2006). Although maintaining the viability of the embryo, it has to be determined whether embryonic stem cell lines derived from a single blastomere that does not compromise the embryo can be considered for clinical studies. Cell Nuclear Transfer (SCNT): Nuclear transfer, also referred to as nuclear cloning, denotes the introduction of a nucleus from an adult donor cell into an enucleated oocyte to generate a cloned embryo (Wilmut et al., 2002).
ESCs differentiation. Differentiation potentiality of human embryonic stem cell lines. Human embryonic stem cell pluripotency is evaluated by the ability of the cells to differentiate into different cell types.
This technique creates ES cells from a single blastomere directly removed from the embryo bypassing the ethical issue of embryo destruction (Klimanskaya et al., 2006). Although maintaining the viability of the embryo, it has to be determined whether embryonic stem cell lines derived from a single blastomere that does not compromise the embryo can be considered for clinical studies.
Nuclear transfer, also referred to as nuclear cloning, denotes the introduction of a nucleus from an adult donor cell into an enucleated oocyte to generate a cloned embryo (Wilmut et al., 2002). The first application of this technique was in 1996 the creation of Dolly the sheep (Campbell et al., 1996). Transferred to the uterus of a female recipient, this embryo has the potential to grow into a clone of the adult donor cell, a process termed “reproductive cloning.” When explanted in culture, this embryo can give rise to embryonic stem cells that have the potential to become any or almost any type of cell present in the adult body. This process is also called “nuclear transplantation therapy” or “therapeutic cloning” because embryonic stem cells derived by nuclear transfer are genetically identical to the donor and thus potentially useful for therapeutic applications. It might substantially improve the treatment of neurodegenerative diseases, blood disorders, or diabetes, whose therapies are currently limited by the availability or immunocompatibility of tissue transplants (Hochedlinger and Jaenisch, 2003). Unfortunately, reproductive cloning is a largely inefficient and error-prone process that results in the failure of most clones during development due both to activation of inadequate pathways of early embryonic development (Solter, 2000) and suppression of pathway of differentiation (Rideout et al., 2001). In contrast, reprogramming errors do not appear to interfere with therapeutic cloning, because the process appears to select for functional cells. Recent advances in the field of nuclear cloning showed that most clones die early in gestation while cloned animals share abnormalities regardless of the type of donor cell or the species used, correlating with aberrant gene expression (Hochedlinger and Jaenisch, 2003). Although experiments in animals showed that by SCNT it is possible to obtain primate ES cells (Byrne et al., 2007) and nuclear cloning combined with gene and cell therapy represents a valid strategy for treating genetic disorders (Rideout et al., 2002), the low efficiency of the technique, the difficulties in obtaining human eggs and the arising ethical problems are significant challenges to the widespread use of SCNT for the production of hESC.
Human embryonic stem cells (hESCs) were first derived from the inner cell mass (ICM) of the blastocyst stage (100–200 cells) of embryos generated by in vitro fertilization (Thomson et al., 1998), but methods have been developed to derive hESCs from the late morula stage (30–40 cells) (Strelchenko et al., 2004), from arrested embryos (16–24 cells incapable of further development) (Zhang et al., 2006) and single blastomeres isolated from 8-cell embryos (Klimanskaya et al., 2006). Because hESCs have the potential to differentiate into normal tissues of all types, the ability to derive and maintain hESCs in culture gave rise to the possibility of having an unlimited supply of normal differentiated cells to engineer diseased tissues to regain normal function (Moon et al., 2006; Skottman et al., 2006).
Nowadays, several studies demonstrating hESC differentiation into specific cell lineages use feeder layers of heterologous cells to maintain hESCs in culture and to signal the hESCs to differentiate into specific cell types (Conrad et al., 2008; Takahashi and Yamanaka, 2006). After transplantation into the recipient, the hESCs and their progeny could be exogenously controlled if they differentiated into malignant cells or if they otherwise grew and/or functioned in an unwanted lineage; if hESCs are to be useful in generating normal tissues for the treatment of human disease, the tissues to be transplanted must be compatible with the host such that the cells derived from the hESCs will not be recognized as “foreign” and rejected as would any transplanted tissue from an unrelated donor.
The first human stem cell line bank was opened in 2004 in the United Kingdom (
In fact for hESCs to be useful for therapy, technologies must be developed to provide them with the specific signals required to differentiate in a controlled lineage, to regulate and/or shut down the growth of hESCs and their progeny once they have been transferred to the recipient. Pluripotency is evidenced by the ability to form teratomas when transplanted in immunodeficient mice, the concern exists that these cells could form malignant tumours in their new host. One strategy for dealing with this problem is to select pure populations of more committed cells for transfer. Demonstrating genetic and epigenetic stability will therefore be important before these cells are used clinically. Moreover attention has to be focused on circumventing the host rejection of transplanted, non-autologous hESC-derived cells (Strulovici et al., 2007).
Therefore, karyotypic abnormalities have been described in several hESC lines, although changes might be at least partially dependent on culture techniques (Mitalipova et al., 2005). In additional to biologic issues directly affecting the stem cell product, it is fundamental that controlled, standardized practices and procedures be followed to maintain the integrity, uniformity, of the human stem cell preparations. Because of stem cells are maintained and expanded in vitro before transplantation, culture conditions compatible with human administration must be used. Feeder cells and sera of animal origin have to be reduced and ideally avoided to reduce the potential risk of contamination by xenogeneic protein. Consequently, life-long immunosuppressive therapy, which can lead to infections and organ-based toxic side effects, such as nephropathy, might be required to prevent graft rejection (Brignier and Gewirtz, 2010).
Cell origins are often defined by one or more cell surface and or intracellular epitopes unique to that particular cell type. Stage-specific embryonic antigen (SSEA) markers are used to distinguish early stages of cell development, denoting pluripotency. These markers are globo-series glycolipids and are recognised by monoclonal antibodies. The SSEA-4 epitope is the globo-series glycolipid GL7. It has been demonstrated that GL7 can react with antibodies to both SSEA-3 and SSEA-4 (Kannagi et al., 1983a; Kannagi et al., 1983b). Human ES cells will express SSEA-3 and -4 during pluripotency and only SSEA-1 upon differentiation (Andrews et al., 1996; Reubinoff et al., 2001; Thomson et al., 1998; Thomson and Marshall, 1998). The TRA-1-60 epitope adheres to a particular epitope of the proteoglycan and is sialidase sensitive, whereas antibody TRA-1-81 reacts with another unknown epitope of the same core proteoglycan molecule. Nanog is a NK-2-type homeodomain gene thought to encode a transcription factor that is critically involved in the self-renewal of stem cells. Thus, it may possibly act to repress genes necessary for differentiation and activate those involved in self-renewal. Lin and colleagues (Lin et al., 2005) demonstrated that the tumour suppressor p53 binds to the promoter of Nanog. Therefore, p53 can stimulate p53-dependent cell-cycle arrest and apoptosis when genetic integrity is not preserved. Oct-4, a POU-domain transcription factor, is highly expressed in ES cells (Niwa, 2001; Reubinoff et al., 2001; Thomson et al., 1998) and has been shown to be essential for maintaining pluripotency (Niwa, 2001). It has been reported that Oct-4 transcripts are nearly exclusively found in pluripotent cells in vivo and within culture. Oct-4 down-regulation is observed in differentiating cells (Rosner et al., 1990). Not only is Oct-4 necessary for the maintenance of pluripotency, but its expression level governs three cell fates once differentiation occurs (Hay et al., 2004; Niwa et al., 2000). Several candidate genes have been reported as targets of Oct-4 based on stem cell expression patterns and immunoprecipitation, but few have been conclusively verified. Target genes of Oct4 include Rex-1, Lefty-1, PDGFalfaR and Utf-1, and those cooperating with Oct4 include Sox2. In the ongoing search for the identification of pluripotent markers, Xu and colleagues have reported that the catalytic component of telomerase, telomerase reverse transcriptase or hTERT, is expressed in undifferentiated cells and down-regulated upon differentiation (Xu et al., 2001).
ES-derived progenitors possess excellent self-renewal and regenerative potential, but the research on these cells is at the beginning. Recently, Jaenisch and collaborators published that the adult cells contain unipotent and multipotent stem cells such as haematopoietic stem cells even if totipotent and pluripotent cells are restricted to the early embryo (Jaenisch and Young, 2008). Although the decrease in developmental potential, the nuclei of most of adult cells maintain nuclear plasticity to reset to an embryonic state. It’s possible to enhance this process by exposing the oocyte to specific factors through nuclear transfer or the cells to pluripotent cell-specific factors by driving over-expression of defined transcription factors. However before the clinical applications of these cells, it’s needed to optimize the engraftment of hESCs and the development of a protocol to obtain similar populations of muscle precursors from human ES cells. ESCs are derived rather easily, and they can grow indefinitely in culture. Second, embryonic stem cells can be manipulated genetically by homologous recombination to correct a genetic defect (Rideout et al., 2001). Recently, Jiang and collaborators demonstrated pluripotency of mesenchymal stem cells derived from adult marrow, differentiating into cells of all three germ layers both in vitro and in vivo after being injected into blastocysts (Jiang et al., 2002). Unfortunately, they did not assess the ability of these cells in correcting a disease phenotype into both human or mouse animal models. Embryonic stem cells can become any type of cell through the use of specific culture conditions or genetic manipulation. To avoid the ethical and practical limitations of therapeutic cloning mentioned above, it would be useful to reprogram somatic cells directly into embryonic stem cells without the use of oocytes. For this reason, it’s necessary to deeply understand the role of several molecular factors in establishing and maintaining pluripotency, such as Oct-4 (Niwa et al., 2002). Oct-4 null embryos cannot form a pluripotent inner cell mass, consequently their development is arrested. To circumvent the need for human oocytes, it could be possible to modify the expression of Oct-4 and its related genes in somatic cells to reprogram their nuclei to an embryonic state.
It is probable that ESCs will suffer from the same acute and chronic rejection problems that accompany other grafts and it is likely that this question will not be answered until these cells are implanted into humans for the first time. Three methods have been proposed to avoid this problem. The first requires the use of somatic cell nuclear transfer (SCNT) techniques, as used to clone animals, to personalize ESCs. The nucleus of a somatic cell from the individual to be treated would be transferred into an enucleated donor oocyte, which would then be used to derive a blastocyst and subsequently isolate ESCs lines that would be genetically identical to the patient (Yang et al., 2007). In this case, any cell generated from the personalized ESC line should not be rejected. However, despite the claims of the South Korean Dr Hwang Woo-suk in 2005 that have subsequently been shown to be fraudulent, SCNT has not been successfully performed in human oocytes. It appears that the process is more complicated in humans than in animals where this technology has been used successfully in many species. The second method makes use of the capacity of ESCs to differentiate into multiple tissues and would involve replacing the recipients immune system with haematopoietic cells generated from the same ESC line as that used for tissue replacement. This technique has been used in solid organ transplants where patients have previously received bone marrow transplantation, and these patients did not require immune suppression (Helg et al., 1994). The third method used genetic manipulation to engineer ESCs in which MHC molecules or other immune effectors have been deleted (Hyslop et al., 2005). All these methods are under development. In addition to the immune response to the cells themselves, animal products are used to isolate ESCs in every methods, and it cause expression of animal proteins on the surface of the ES cells. This will also induce an immune response and a large amount of work is currently going into deriving and maintaining ESCs in total absence of these animal products and also undertaking these processes to the good manufacturing practice (GMP) standards required for clinical use. It is in the development of GMP protocols for the derivation and manipulation of ES cells.
In addition to the problems connected with immune interactions, there are other important problems in providing a suitable number of cells for transplantation. The first problem is related to have the very large numbers of cells required for tissue replacement without tumour formation. As previously discussed, ES cells can form teratomas and therefore all undifferentiated cells will need to be removed from a graft. It is possible to circumvent this problem genetically modifying the cells by a suicide gene system. The expression of the suicide gene could be driven by the promoter of a gene such as Oct4 that is only expressed in undifferentiated cells; this technology has been used in gene therapy systems. Activation of the suicide gene by drug treatment of cell cultures would give rise to the death of the undifferentiated cells not affecting differentiating cells. Other methods include the expression of a genetic label such as the green fluorescent protein or a marker that enables cell sorting by FACS (Strulovici et al., 2007). In this case, cells could be selected using FACS for the surface markers expressed either excluding undifferentiated cells or positively selecting for the required differentiated cell type. Even if contaminating undifferentiated cells are removed, remain the problem due to the necessity to have a good number of mature cells able to form a robust graft and also maintaining the cells where they have to perform their therapeutically action. Differentiation protocols are being studying to generate very large number of cells (Joannides et al., 2007) and scaffolds able to keep the cells in place (Ferreira et al., 2007). Another aspect of tissue replacement that cannot be ignored is that few tissues are formed from a single uniform cell type. In order to regenerate a functional organ, it will be necessary to develop other structures such as vasculature and lymphatic drainage systems as well as complex mixed cell populations (Caspi et al., 2007). These problems present a major challenge to tissue culture technology not only for ESCs but also for adult stem cells, and the development of new systems will be important in order to utilize ES cells to their maximum potential.
The use of hESCs in medical research has focused much attention from many sectors of the public. Religious, historical, cultural, medical, and other points of view have contributed to a very vigorous and wide-ranging discourse over the use of these materials (Leist et al., 2008). Some consider research with hESCs to be inherently immoral because these individual’s believe that life begins with fertilization of the ovum, and the destruction of an embryo with the potential to develop into a viable human being is thought like an infanticide. For this reason, the American federal government severely restricted access and use of hESCs in 2001. These restrictions have now been largely overturned by the Obama administration. In contrast, proponents of this line of research insist that the potential benefits to human from this research mitigate such concerns. They also argue that hESCs are made from unwanted fertilized ovum that would likely be destroyed in any event. Stem cells created by means of nuclear transfer share the same ethical concerns. Furthermore, because these cells have the potential to generate a complete embryo, they also raise the even more highly charged possibility of cloning human beings, so-called reproductive cloning (Brignier and Gewirtz, 2010). Many organizations and countries have already banned reproductive cloning of human beings. Because this procedure can be used to generate stem cells for therapeutic purposes, in countries where this type of cloning is legal, such as Australia and the United Kingdom, the created embryos must be destroyed within 14 days. A human nucleus is transferred into an animal’s oocyte, creating a hybrid embryo that must be destroyed within 2 weeks and cannot be implanted. Clearly, creation of such tissues raises even more complex issues. Finally, the issue of financial compensation for embryo and gamete donors is also controversial, with guidelines for this problem being proposed by the International Society of Stem Cell Research (
Among the important potential applications of gene therapy to hESCs is the correction of genetic diseases. Although many hereditary disorders can be targeted by gene therapy vectors alone, the combination of gene therapy and stem cell therapy may have added utility, where cells differentiated from hESCs would act as factories to produce therapeutic proteins or where a high proportion of corrected cells could be developed. In the case of circulating proteins (e.g., factor IX, factor VIII, von Willebrand factor, α1-antitrypsin), it may be possible to establish tissue reservoirs distant from the normal site of the secreted product (Mountford, 2008). The cells differentiated from hESCs can be delivered to a site accessible and receptive to transplantation, even if that tissue is not the normal site of production of the protein of interest. When the product is not secreted, the hESCs with their regulated genetic characteristics must be differentiated to the correct cell type (e.g., the cystic fibrosis transmembrane conductance regulator for cystic fibrosis or dystrophin for muscular dystrophy). Such proteins can exert their influence only at the appropriate site, and there is no known mechanism by which cells expressing the protein remote from the affected tissue could have a therapeutic effect. Additional drawbacks are to be solved in order to obtain the successful therapeutic application of gene-modified hESCs including whether the hESC themselves, or the expressed product, will be toxic or immunogenic in the recipient. If the recipient of the cells, never exposed to the protein before, as in deletion or nonsense mutants, can be showed an immune reaction against the protein, limiting the effectiveness of the therapy (Mountford, 2008).
To develop hESC-based therapies, it is obvious that strategies capable of mitigating risks related to the therapeutic use of hESCs should be pursued the development of the therapies. These strategies might include several non–mutually exclusive mechanism for ablating all genetically modified cells while sparing most endogenous cells. The introduction of a step in the development of the therapy at which a single genetically modified cell would be isolated, expanded, and characterized with respect to the location of the mutation would allow an analysis of the relative risk of the insertion site. Similar limiting dilution cloning strategies are now routinely performed during the original isolation of a stem cell line to ensure that only one karyotype is represented (Mountford, 2008). Progress in understanding how insertional mutagenesis can lead to uncontrolled growth of stem cells is an essential prerequisite for this analysis and is currently an active area of research. Genetic modification can be used to enhance our ability to conduct such an isolation step by adding a convenient ligand for cell isolation. Genetic modification is also potentially useful for solving the problem of uncontrolled cell growth. Incorporating the genes for an ablation strategy at the same time as the genes for the therapeutic strategy would give the best chance of ensuring that the safety mechanism will be present when and if needed. Initial applications of genetically modified hESCs are likely to occur where the risk/benefit ratio tilts in favour of benefit, as in fatal disorders for which there is no therapy. The risks of the hESC therapies will have to be understood and probably reduced to maintain an appropriate risk/benefit ratio before these technologies can be applied to diseases that are inherently less dangerous to the patient. Gene therapy should prove to be valuable in reducing the risks associated with making hESC therapy a reality (Mountford, 2008).
In particular, the ability to generate cells with in vivo muscle regenerative potential in culture and systemically transfer them to recipients is an important step towards the therapeutic application of ES cell-derived cells. Unfortunately, it’s not still provided a reproducible method to generate ES-derived myogenic progenitors for skeletal muscle regeneration. An ES cell–based therapy would have many advantages: it could allow the transplantation of a more primitive cell with greater replicative potential and patient-specific ES cells could be induced from adult somatic cells. Moreover, the derivation of an ES cell–derived myogenic population with proliferative and regenerative potential has not been accomplished. Only two papers described some evidences for engraftment on transplantation of an ES cell–derived population but they were limited to qualitative detection of donor derived cells in recipient muscle (Bhagavati and Xu, 2005; Kamochi et al., 2006). The Pax3 ES cell–derived population exhibited good potential for skeletal muscle regeneration but several studies concerning their capacity of replenish satellite cells-niche are in progress (Darabi et al., 2008a). On the other hand there is enough optimism about the ESC-based therapies because of it may offer reliable and cost-effective therapeutic substitute for treatment of neuromuscular disorders as DMD or BMD. Moreover there are critical issues that need attention in case of ESC-based approaches. No enough knowledge there are about genetic and epigenetic stability of hESC lines over longer time periods and it’s not negligible the possible uncontrolled cell proliferation and reprogramming of ESCs in vitro. The injection of these cells could probably generate an immune rejection needed an immunosuppressive therapy. Optimization of a generic differentiation protocol and its empirical testing with a better understanding of the molecular processes governing ESC differentiation can guarantee the clinical use of these cells. In conclusion the success of the clinical application of adult or embryonic stem cells will be employed to a large-scale production of desired cell type with appropriate functionality, an optimal number of cells for transplant, a modification of less invasive delivery systems and a technique to label cells for transplant and subsequent tracking of cell fate.
The major impediment to ES based therapies in humans involves the moral and ethical problems linked to the blastocyst destruction and oocyte donation necessary to generate patient-specific pluripotent stem cell lines. These limitations have encouraged researchers to understand the mechanisms regulating pluripotency and to experimentally determine its gene expression program. Recent works describe the derivation of ES-like iPS cells from adult mouse and human cells (Nakagawa et al., 2008; Takahashi et al., 2007; Yu et al., 2009) by introducing specific sets of genes encoding transcription factors expressed in undifferentiated ES cells to reprogram the adult cells. Although initial studies indicating these cells to share characteristics of ‘‘true’’ ES cells, more detailed work is needed to determine how closely they resemble ES cells. Like ESCs, iPS cells can differentiate into all adult cell types. Researchers now have the ability to create tissue-based models of human disease based on cells derived from individual patients. This technology has the potential to herald a new era of patient-specific, cell-based medicine; however, given the oncogenic potential of undifferentiated iPS cells due to the unsafe reintroduction of these genes (Takahashi and Yamanaka, 2006), the safety of these cells has to be tested accurately before attempting any therapies. It has been demonstrated that continuous over-expression of transcription factors, especially the c-myc oncogene, may be associated with tumorigenesis (Takahashi et al., 2007). Even if it was demonstrated that the promoters of these viral vectors can be silenced by endogenous gene expression during reprogramming, chimeric mice derived from iPS cells were showed to be more prone to tumour formation. Following ameliorations in iPS technology, Nakagawa and co-workers generated pluripotent stem cells without c-myc over-expression both from mouse and human fibroblasts, with lower efficiency (Nakagawa et al., 2008). In addition, chimeric mice created from these non-myc iPS cells do not form tumours at an elevated rate. Recently, Chuang et al. proposed the use of baculoviral systems as a new gene delivery vector for stem cell engineering, and in particular for transgenic expression in human ESCs (Chuang et al., 2007). These vectors can be used for large segments, more than 30 kb, that do not fit into adenoviral or lentiviral vectors and could limit the risk derived from the great immunogenicity of adenoviral vectors.
In 2006, Yamanaka et al. identified four transcription factors—Klf4, Sox2, Oct4, and c-Myc able to transform mouse fibroblasts in pluripotent clones through retroviral transduction. The clones were selected for their ability to reactivate the non essential Oct4 downstream target gene, Fbx15 (Takahashi and Yamanaka, 2006). This first-generation of
Generation of iPS cells. Reprogramming of adult stem cells in iPS cells mediated by Oct-4, Klf4, Sox2 and c-Myc give raise to ES like cells with embryonic potential.
iPS cells exhibited partial demethylation and reactivation of the canonical pluripotency governing genes Oct4 and Nanog. In 2007, fully pluripotent iPS cells were generated by increasing the stringency of the selection strategy and selecting for reactivation of the pluripotency regulators Oct4 and Nanog themselves (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007). These new second-generation iPS cells had fully demethylated Oct4 and Nanog promoters. These cells could not form viable mice by tetraploid complementation; an assay in which ES or iPS cells are injected into tetraploid blastocysts, resulting in embryos derived entirely from the injected cells, while extraembryonic tissues are derived from the host blastocyst. It is controversial if this failure is due to effects of the randomly integrated retroviral vectors used for reprogramming or represents a more fundamental defect in the developmental potency of iPS cells. Pluripotent iPS cells could be identified on the basis of their morphology, eliminating the need for genetically modified reporter genes and permitting the isolation of iPS cells derived from human fibroblasts (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2009). After the work of Yamanaka, Yu et al generated iPS cells starting from a combination of Oct4, Sox2, Nanog, and LIN28. The finding that direct epigenetic reprogramming with different combinations of transcription factors can be applied to human cells represents the breaking of a species barrier that SCNT has yet to overcome. The therapeutic value of iPS cells is the presence of proviral integrations harboring known oncogenes, particularly c-Myc, as well as Oct4 and Klf4. c-Myc was dispensable for iPS generation from fibroblast target cells (although iPS formation occurred with reduced efficiency), and chimeric mice derived from three-factor iPS cells (Oct4, Sox2, and Klf4) did not exhibit tumour formation while the cells derived from the first four-factors showed tumorigenic characteristics (Nakagawa et al., 2008; Wernig et al., 2007). Despite the non essential role of c-Myc in the reprogramming process, the potential for insertional mutagenesis and the oncogenic properties of the other reprogramming factors has prompted several groups to undertake direct epigenetic reprogramming approaches using either nonviral methods, or retrospectively eliminate proviral integrations after iPS cell generation.
Different reasons have drive the researchers to evolve strategies for lentivirus-free iPS cell generation. First, the introduction of foreign DNA able to integrate in random positions in the genome represent a risk for cell physiology giving rise to an insertional mutagenesis. The foreign DNA could destroy reading frames of genes or influences gene regulation. Moreover silencing of lentiviral transgenes is incomplete in iPS cells leading to reactivation of lentiviral vectors (Wernig et al., 2007). In fact basal expression of lentiviral reprogramming factors is found even in fully reprogrammed cells (Hotta and Ellis, 2008; Pfannkuche et al.). Pfannkuche et al. demonstrated that in murine iPS cultures, there was different expression of viral Oct4 by three orders of magnitude between spontaneously differentiating iPS and stable subclones of the same origin (Pfannkuche et al.). From several studies, it is known that altered levels of pluripotency factors influence the fate of pluripotent cells. Overexpression of Oct4 seems to direct ES cells towards an endodermal fate (Niwa et al., 2000; Pesce and Scholer, 2001). More strikingly, elevated levels of Sox2 can alter the whole network of pluripotency factors and abolish maintenance of pluripotency (Kopp et al., 2008; Rizzino, 2009). The best strategy to eliminate reprogramming factors or to prevent integration of foreign transgenes for generation of iPS cells for therapeutic issues could be to employ techniques that are able to reprogram somatic cells without the use of any type of DNA. One possibility to achieve this aim is the use of cell-permeable transcription factors. The application of cell permeable proteins for direct reprogramming has been shown with human newborn fibroblasts, but, again, the efficiency of reprogramming appears low (Kim et al., 2009a). Kim and co-workers includes incubation with the cell permeable factors for 42 days with six passage steps during incubation. Even if the efficiency appears low, this study demonstrated for the first time that cell-permeable proteins can reprogram somatic cells. However, the long incubation time of 6 weeks in the presence of recombinant reprogramming factors in culture media, could be difficult. The application of cell-permeable reprogramming factors is a very promising approach that certainly justifies further investigation to achieve efficient, less time consuming reprogramming of cells from adult donors. Recent results suggest that a substantial amount of cell permeable reprogramming factors is enclosed in endosomes after cell uptake and might therefore be unavailable for reprogramming (Pan et al.). Another possibility to perform reprogramming is the introduction of RNA that can be transfected into cells. Transport of synthetic mRNAs to the nucleus is not necessary and, therefore, the transfection efficiency to deliver mRNA to the cytoplasm might be high. The stability of RNA limit the reprogramming efficiency and to obtain the right amount of protein translated it will be necessary several rounds of transfection to achieve reprogramming. A further strategy to induce pluripotency without the generation of stably transfected cells is the use of non integrating viruses. Adenoviral vectors were designed to express reprogramming factors and transport the coding sequence transiently into the target cells. Application of the adenoviral approach on fetal human fibroblasts resulted in the generation of stable human iPS cells without viral integration. The human iPS cells form teratomas in vivo, which is the most stringent assay applicable to test their pluripotency (Zhou and Freed, 2009). Adenoviral vectors could enhance the production of virus- free iPS cells but the use of viruses that are DNA-based still bears a residual risk of integration into the host genome. This might be overcome with an RNA-based virus to generate safe iPS cells without application of DNA based vectors.
Another possible approach is the use of a single plasmid containing Oct4, Klf4, c-myc and Sox2 stably transfected into somatic cells for reprogramming. The plasmid expressed a single mRNA that codes for all four reprogramming factors and the red-fluorescent protein (RFP) in order to discriminate the transfected cells (Kaji et al., 2009). Different studies are also focused on the application of transposon-based systems. The application of the piggy-BAC transposon allows delivery of reprogramming factors into cells of different organisms and stable integration of the reprogramming genes into the genome by action of the transposase enzyme transiently transfected in a plasmid, and it is able to catalyse the integration of the reprogramming factors (Woltjen et al., 2009). The whole cassette is flanked by inverted terminal repeats, which mediate insertion into the genome by the host-factor independent activity of a transposase. The principal advantage of the system is not only the higher efficiency to deliver reprogramming factors but also the possibility to remove the transgene. Expression of reprogramming factors from the transposon was controlled by a doxicyclin inducible promoter, in order to guarantee a temporal control of transcription factors.
Researchers are looking for solutions to reduce the number of factors needed for reprogramming. One of the reasons is to reprogram the cells using chemical compounds in order to standardize the procedure conditions, to control the response of each compound and to regulate the reprogramming factor expression. Another reason is that reduction of reprogramming factors to perhaps a single transgene could guarantee the understanding of mechanisms underlying reprogramming. Between the four factors used by Yamanaka, c-myc has been thought to have the highest oncogenic potential. The three other factors were not associated with tumorigenesis. The role of Klf4 in carcinogenesis is ambiguous. It can act as a tumour suppressor gene, especially in gastrointestinal tumours, or as oncogene, in the development of breast carcinoma where it is involved in the early phase of malignant transformation (Rowland and Peeper, 2006). The first reduction to three factors Oct4, Sox2 and Klf4 induced pluripotency in mouse and human fibroblasts (Nakagawa et al., 2008; Wernig et al., 2007) and other somatic cell types (Park et al., 2008). Moreover the generation of iPS cells was possible combinating Oct4, Sox2 and c-myc in the absence of Klf4. It was concluded that Oct4 and Sox2 in combination with either c-myc or Klf4 were sufficient (Park et al., 2008). Other researchers studied the use of another set of factors including Sox2, Oct4 and the nuclear receptor Essrb, which could replace Klf4 for reprogramming (Feng et al., 2009). However, the estimated reprogramming efficiency was ten times higher than in the original 4-factor approach in fibroblasts. Finally, reprogramming using only Oct4 in the absence of any other factor became possible in neural stem cells derived from adult mice (Kim et al., 2009b) and humans (Kim et al., 2009a). Another strategy is the substitution of single reprogramming factors by small molecules. For example c-myc or Klf4, could be substitute with valproic acid (VPA), thus making reprogramming of human fibroblasts possible only by retroviral transduction of Oct4 and Sox2 (Huangfu et al., 2008), with a 200-fold reduction of reprogramming efficiency compared to Oct4, Sox2 and Klf4 in combination with VPA. VPA acts as histone deacetylase inhibitor (Huangfu et al., 2008). Other authors utilized high-throughput assays to identify chemical factors which can substitute reprogramming genes. For example, Klf4 could be chemically substituted by the application of kenpaullone (Lyssiotis et al., 2009), and two other small molecules (BIX-01294 and Bayk8644) could enable reprogramming of embryonic fibroblasts with only Oct4 and Klf4.
Due to the low efficiency of iPS production, different factors have been investigated in order to improve this procedure. Simultaneous transduction of c-myc, Oct4, Sox, Klf4, with Nanog and Lin28 increases efficiency dramatically (Liao et al., 2008). Other studies have shown that expression of p53 siRNA and UTF1 dramatically increases iPS colonies (Zhao et al., 2008). While p53 siRNA remarkably increases the number of iPS colonies, UTF1, a pluripotent marker and necessary for the maintenance of pluripotency in mES cells (Gaspar-Maia et al., 2009). By affecting different pathways, these two factors together can synergize iPS generation (Zhao et al., 2008). Other pathways which have an impact on iPS cells formation include the TGFβ and the MEK-ERK pathway. The role of these pathways in cell survival has made them beneficial in reprogramming studies. Chemical inhibitors of the MEK pathway apparently inhibit the growth of non-iPS cells while increasing the growth rate in reprogrammed iPS cells. Suppressing MEK and TGFβ pathways concurrently with chemical compounds increases iPS amounts. This study showed that combined suppression of both pathways resulted in extensive amounts of iPS generation in comparison to individual inhibition of each pathway (Lin et al., 2009). Repression of the Ink4/Arf locus which is a regulator of the p53 p21 pathway has a positive effect on reprogramming. Different pluripotency genes are controlled by the expression of different micro RNAs expressed in the cell. For example, let-7 is one of the miRNAs which targets the 3’UTR and ORF’s of several pluripotency expressed genes including c-myc, Oct4, Sox2 and Nanog. Suppressing let-7 by antisense inhibitors improves iPS generation several fold in mouse embryonic fibroblasts (MEFs) (Melton et al., 2010). Reduced oxygen levels favour the growth of haematopoietic stem cells and maintain ES cells in a pluripotent state. Induction of pluripotency in hypoxic conditions increases the number of iPS colonies and vitamin C elevates iPS reprogramming efficiency (Esteban et al., 2010). The increase in iPS formation is not due to its antioxidant activity but vitamin C inhibits p53, thus facilitating the induction of iPS cells (Shi et al., 2010). Vitamin C extends the life span of both iPS and MEF cells pointing at roles for vitamin C in anti-ageing. Composition of the iPS culture medium also defines the efficiency of iPS formation. The use of transcription factors together with different chemical agents can enhance iPS induction. This can result in the derivation of more pluripotent cells in a shorter time, making its application more convenient for clinical purposes in a not too far future.
Following the success of haematopoietic stem cell therapy in the treatment of haematological diseases, the potential application of cell based therapy has been extended to the treatment of other human diseases. In particular, different types of adult stem cells, including bone marrow, peripheral haematopoietic, and mesenchymal stem cells (MSC) have been evaluated in the treatment of different diseases ( Charwat et al., 2010; Siu et al., 2010). ESCs have been explored for tissue regeneration because of their ability to differentiate into various therapeutic relevant cell types in\n\t\t\t\t\tvitro (Murry and Keller, 2008). Despite this, there is limited progress in the use of ESCs for tissue regeneration in humans due to various technical, social and religious issues (Kiskinis and Eggan). The generation of patient-specific iPS cells has the advantage of avoiding many of the ethical concerns associated with the use of embryonic or foetal material, and have no risk of immune rejection. Currently, several therapeutic relevant cell types, including motor neuron (Dimos et al., 2008), hepatocytes (Song et al., 2009), pancreatic insulin producing cells (Zhang et al., 2006), haematopoietic cells (Hanna et al., 2007), retinal cells (Carr et al., 2009), cardiomyocytes (Zwi et al., 2009) and MSCs (Lian et al., 2010), have been successfully derived from human iPS cells, and some of them have been tested to treat diseases in animal models. The use of iPS cells has thus been proposed as diagnostic and therapeutic tools for different haematological disorders (Ye et al., 2009). Nelson et al. (Nelson et al., 2009) reported the use of iPS cells for myocardial repair in animal models of acute myocardial infarction. There are several major challenges to overcome before iPS cell technology is applied in clinical practice. Now, current iPS cells are not “clinical grade”. Genome-integrating viral vectors used for reprogramming are known oncogenes, particularly c-Myc, Oct4 and Klf4, such that iPS cells thus generated are unlikely to be safe for clinical application. Nonetheless recent technological advances, including reprogramming without viral integration such as plasmids or direct reprogramming protein delivery assays can solve this problem (Kiskinis and Eggan, 2010; Saha and Jaenisch, 2009). Despite the challenges in the therapeutic use of iPS cells, preclinical studies have provided the proof-of-concept that patient-specific iPS cells can provide an unlimited cell source to produce massive therapeutic cell types, such as cardiomyocytes and MSCs, and can be prepared in an “off the- shelf ” format for cell transplantation.
Given the many potential risks of applying autologous iPS cell treatment to human subjects, iPS cell therapies may encounter strict regulatory restrictions. For instance, it took Geron Corporation more than 6 years to receive approval from the Food and Drug Administration (FDA) for its human ES cell-derived neuronal cell (GRNOPC1) therapies in terms of cell product safety and reliability. Recently, a second company has presented an investigational new drug for a phase I/II trial using human ES cell-derived retinal pigment epithelial (RPE) cells to treat patients with Stargardt’s Macular Dystrophy (SMD), one of the most common causes of juvenile blindness. The sponsoring company, Advanced Cell Technology (ACT), has performed years of testing to show that differentiated RPE cells can improve the visual performance of rats without adverse effects (e.g., teratomas) in hundreds of treated animals. Another issue that may hinder the clinical translation of iPS cell therapies is the economic feasibility of producing individualized iPS cell therapeutic products. The viability of a business model for patient-specific iPS treatment is still unknown. It may well be the case that few if any pharmaceutical companies will be able to produce cost-effective individualized iPS cell products tailored for a single patient at a time. To be commercially feasible, these cells will need to be made in standardized, large-scale production, and the individual needs or profiles of patients will need to be easily assessed to allow matching and wide distribution (Sun et al., 2010). Several groups have also begun the generation of patient-specific human iPS cell lines. Park et al. generated a library of patient-derived iPS lines from numerous disorders including Huntington’s disease, juvenile diabetes mellitus, Down syndrome, muscular dystrophy, and several others (Park et al., 2008). Of particular interest are iPS cells derived from neurodegenerative diseases (Dimos et al., 2008; Ebert et al., 2009). These iPS lines can be differentiated in vitro into the affected neuronal cell type, generating for the first time a model for idiopathic neurodegenerative disorders which can be screened in culture for the onset, cell autonomy, and contribution of environmental factors to the phenotype. Ultimately, if human neurodegenerative phenotypes can be recapitulated in iPS cell-derived culture models, these cells could be screened using chemical libraries to identify molecules that can arrest or even reverse the progression of these disorders.
The comparison of iPS and ES cells revealed that these cells are very similar. The differentiation capacity of iPS cells seems to resemble that of ES cells; iPS derived somatic cells are comparable to those derived from ES cells. Several studies describe the derivation of a variety of cell types from murine and human iPS cells, among them cardiomyocytes, smooth muscle cells, hepatic cells and neurons with similar differentiation behaviour of iPS and ES cells. The transcriptome of iPS and ES cells was analyzed by Gene chip analysis; the results showed that these cells are very similar but they are not identical. Chin and co-workers compared the expression pattern of different human ES and iPS cells; they analysed histone modifications and the expression of non-coding miRNAs in both type of cells and they constructed a fingerprint of iPS cells that distinguishes them from ES pluripotent cells (Pfannkuche et al., 2010). The comparison of the transcriptome of early and late passage iPS cells with ES cells revealed two datasets of differentially regulated genes. They identified a subset of 318 genes differentially expressed between human ES and iPS cells at any stage. The genes that are higher expressed in iPS cells were also found higher expressed in fibroblasts than in ES cells. The same conclusion is valid for genes that were expressed at a lower level in iPS than ES cells are usually also lower expressed in fibroblasts than ES cells. Together, these findings point at an imperfect reprogramming of a small set of genes. It is not known if there are implications of iPS fingerprint for the physiology of these cells. It is important determine the genes that are differentially regulated and that could influence the cellular physiology. It is likely that some of these gene functions are redundant with others that are not affected by incomplete reprogramming and, therefore, do not influence cell physiology (Pfannkuche et al., 2010).
Beside genes that constitute a potential iPS cell fingerprint, there are varying gene sets that are differentially expressed in individual iPS cell lines. It will be interesting to see if properties of the ancestral cell types are transmitted to the iPS cell line generated. In this regard, partial reprogramming plays a role and one fascinating aspect to address is if partial reprogramming alters the differentiation capacity of a cell in a way that it potentially influences the fate decision of the partially reprogrammed iPS cells. The differences between iPS and ES cells could be an assays to measure the quality of iPS cells. Although it has been shown that the overall gene expression of iPS cells differs from normal ES cells, this comparison has never been made between cells from the same individual. Usually iPS cells are compared with those ES cells either derived from another species or from a different individual; raising concerns about whether these are informative approaches. It is clear, therefore, that iPS cells derived from the trophoblast of an embryo compared with ES cells derived from the inner cell mass of the same embryo would give a more explicit view of how distinct or similar these cells really are (Pfannkuche et al., 2010).
Although ESCs and iPS cells are now the most studied cells for clinical applications in neuromuscular diseases, different stem cells isolated from adult tissues was extensively used – and are still used – unfortunately with poor results. For several years after they were discovered, the satellite cell were considered as the only cells responsible for the growth and maintenance of skeletal muscle. With the improvements of cell-isolation technology, a number of markers were described to identify muscular and non-muscular subpopulations able to actively participate in myogenesis (Meregalli et al., 2010). In the skeletal muscle, adult multi-lineage progenitor cell populations were showed to have myogenic potential, such as muscle-derived stem cells (MDSCs) and muscle-derived CD133+ progenitors. Moreover, it was also shown that non-muscular resident stem cells could participate in myogenesis (Krause et al., 2001; Mezey et al., 2000; Pittenger et al., 1999; Prockop, 1997). In particular, a subpopulation of CD133+ cells was isolated form the blood, playing an important role in myogenic development (Torrente et al., 2003b) while mesoangioblasts were identified in the dorsal aorta of avian and mammalian species (Cossu and Bianco, 2003).
Satellite cells are small progenitor cells that lie between the basement membrane and sarcolemma of individual muscle fibers: normally they are quiescent, consequently they cannot differentiate nor undergo cell division. Oxidative stress and specific stimuli form the environment can activate them, so that they differentiate, proliferate as skeletal myoblasts and activate myogenic differentiation to form new myofibers. Recently, Montarras and colleagues were able to directly isolate a pure population of satellite cells from diaphragm muscle of a Pax3-GFP knock-in mouse (Montarras et al., 2005). After FACS and gene expression analysis, they purified a predominantly quiescent population of satellite cells expressing Pax3, CD34 and Pax7. These cells were firstly injected into dystrophic dogs and restored dystrophin expression 3 weeks post-transplantation. Transplanted into irradiated dystrophic mice, they also formed a small amount of the satellite cell pool that expressed both Pax7 and Pax3 (Montarras et al., 2005). If compared with the results obtained after the injection of human cells isolated from adult muscle, these cells showed an incredibly efficient level of muscular regeneration (Morgan et al., 1996). Since they were doubtless highly myogenic, satellite cells were not considered in a clinical point of view, because it was difficult to isolate them and above all to proliferate and expand them to obtain the right number for transplantation experiments. Moreover, the growth of freshly isolated satellite cells in vitro significantly reduced their in vivo myogenic potential.
Muscle-derived stem cells (MDSCs) are a recently-identified subpopulation of cells that resides within skeletal muscle and possess the ability to self renew and to differentiate into other mesodermal cell types (Sarig et al., 2006; Tamaki et al., 2007). Furthermore, it’s known that these cells are distinct from satellite cells (Asakura and Rudnicki, 2002; Qu-Petersen et al., 2002) and that, when appropriately stimulated, they could preserve their myogenic potential in vitro even after differentiation into other lineages (Negroni et al., 2006). In the last years, different works assessed the capacity of MDSCs to differentiate and regenerate skeletal muscle when transplanted into animal models. Sca-1+CD34+ stem cells were purified from the muscle tissues of newborn mice, showing multipotency in vitro. Moreover, after intra-arterial injection, these cells were able to interact and firmly adhere to endothelium in mdx muscles microcirculation and then participated in muscle regeneration (Torrente et al., 2003a). Qu-Petersen and collaborators isolated a MDSCs population Sca-1+/-CD34+/-c-kit-CD45- and demonstrated that they displayed a better transplantation efficiency than satellite cells (Qu-Petersen et al., 2002). MDSCs were also identified in human muscle, expressing in proliferation the CD133 antigen and also desmin and α-SMA when cultured in myogenic conditions (Miraglia et al., 1997). Moreover, among human MDSCs, it was identified a subpopulation of progenitor stem cells with neurogenic properties (Alessandri et al., 2004). According to these evidences, MDSCs are suitable for clinical perspectives as they are easy to proliferate, migrate through the vasculature, and are multipotent, although it could be necessary to better investigate their physiological location (Deasy et al., 2005; Deasy et al., 2001).
Mesoangioblasts are multipotent progenitors of mesodermal tissues, physically associated with the embryonic dorsal aorta in avian and mammalian species, expressing Flk-1, stem cell antigen 1, CD34 and various leukocyte molecules (Cossu and Bianco, 2003; Tagliafico et al., 2004). It was shown that mesoangioblasts treated with a lentiviral vector expressing human microdystrophin were able to produce dystrophin-positive myofibers after injection in animal model of DMD and ameliorated muscle function and mobility (Cossu and Sampaolesi, 2007; Sampaolesi et al., 2006). Furthermore, to improve their efficiency of muscle repair, mesoangioblasts were treated to increase their migration to skeletal muscle and to reduce unspecific trapping in the capillary filters of the body, such as liver and lung (Galvez et al., 2006).
A role for CD133 as a marker of stem cells with the capacity to engraft and differentiate to form functional non-haematopoietic adult lineages and contribute to disease amelioration via tissue regeneration emerged in the last years. Human CD133+ cells, isolated from peripheral blood and manipulated in vitro to undergo myogenesis, were shown to ameliorate disease via a direct contribution to muscular regeneration when transplanted into dystrophic mice (Torrente et al., 2004). In particular, they restored dystrophin expression and eventually regenerate the murine satellite cells pool after intramuscular and intra-arterial delivery. Human CD133+ cells colonized the mouse muscle and formed hybrid regenerated fibers expressing human dystrophin. Moreover, they were detected in several vessels near areas of regeneration, where they expressed human ve-cadherin and CD31 (Torrente et al., 2004). A CD133+ stem cell subpopulation was also identified in normal and dystrophic muscles. They were positive for CD45 antigen, indicating their hematopoietic commitment while the expression of Pax-7, Myf-5, MyoD, m-cadherin, MRF-4, and myogenin after 24 days of culture in the proliferation medium and their ability to differentiate into multinucleated myotubes expressing MyHCs suggested a myogenic commitment (Torrente et al., 2007). According to these data, CD133+ stem cells were considered as a possible tool in the treatment of degenerating diseases. Stamm and collaborators showed that transplanted BM-derived CD133+ cells improved function of infracted myocardium probably as a result of the amelioration in blood vessel formation (Stamm et al., 2003) while Torrente and co-workers demonstrated that intramuscular transplantation of muscle-derived CD133+ cells in DMD patients was a safe procedure and feasible. DMD patients showed an increased number of capillaries per muscle fiber and expressed a change in the ratio of slow-to-fast myosin myofibers (Torrente et al., 2007). Human dystrophic blood- and muscle-derived CD133+ expressed an exon-skipped version of human dystrophin after transduction with a lentivirus carrying a construct designed to skip exon 51 and participated in vivo in muscle regeneration (Benchaouir et al., 2007). This combination of cell- and gene-based approaches via the ex vivo introduction of corrective genes into dystrophic CD133+ cells permitted – in a clinical point of view- the use of patient’s own cells: autologous transplantation would reduce the risk of implant rejection.
Several questions remain to be answered before any of the previously described cell preparations can be moved into clinical trials even if there has been great advance in the generation of cell populations showed in vivo myogenic potential. Based on their unique characteristics and in vivo skeletal muscle regeneration potential, adult stem cell populations discussed in this review are excellent clinical candidates. As shown before, mesoangioblasts (Cossu and Bianco, 2003) and blood derived CD133+ (Gavina et al., 2006) have the ability to migrate through the vasculature, most do not. Potential future methods to increase the migratory ability of stem cell population include the identification of cell surface markers like adhesion molecules (Torrente et al., 2003b) and appropriate growth factors (Horsley et al., 2003; Torrente et al., 2003b). Mesoangioblasts serve as a paradigm for widespread distribution, and after treatment with growth factors are able to correct efficiently the dystrophic phenotype. For now the intra-arterial injection of mesoangioblasts represent a hope for patients suffering from various muscular dystrophies. Satellite cells was one of the first cell types used in cell-based therapy of muscular dystrophy. Expanded satellite cells or myoblasts were isolated from wild-type mice and intramuscularly injected in dystrophic mdx mice (Conway et al., 1997; Tremblay et al., 1998); unfortunately it was demonstrated that myoblast transplantation is an inefficient technique because of the low efficiency of the dystrophin production in muscle fibers of DMD patients and no functional or clinical improvement in the children (Peault et al., 2007). In possible future clinical trials, adult stem cells purified from patients suffering for neuromuscular disorders could be ex vivo engineered and re-injected in the initial donor intra-arterially. The intra-arterial injections of the patient’s own stem cells transduced allow the distribution of the cells to the whole body musculature so that it could be possible to take care of severe-affected patients that have reduced mass body, as in DMD and BDM pathology (Brignier and Gewirtz, 2010). One of the most important problem to solve for future clinical application is the amelioration in safety procedures of gene’s modifications. One of the most reliable methods for gene therapy, fully utilized in DMD clinical approaches, seems to be the exon skipping mediated by AONs or molecules like PTC124. Ongoing phase I/II studies try to assess the efficacy and the safety of intramuscular administered morpholino oligomer directed against exon 51 (AVI-4658 PMO). Morpholinos can interfere with mRNA splicing processes by preventing the formation of the snRNP complex or by interfering with the binding sites for other regulatory proteins (Vetrini et al., 2006). They mediate the exclusion of exons from the mature mRNA as AONs. PTC124 was shown to partially restore dystrophin production in animals with DMD due to a nonsense mutation. The main purpose of a phase II study completed on May 2007 was to understand whether PTC124 can safely increase functional dystrophin protein in the muscles of patients with DMD due to a nonsense mutations. This study demonstrated the safety and the efficacy of the PTC124 treatment; now three ongoing phase 2a and 2b studies are started in DMD and BMD patients (www.clinicaltrials.gov).
A decade of studies in human ESCs has yielded remarkable progress and understanding in stem cell biology. The technical challenge of creating patient-specific ESCs, the ethical issues arising from the foetal origin of human ESCs and the potential risk of immune rejection make broad clinical application of this cell type difficult. Recent advances in human iPS cell technology can potentially circumvent these disadvantages: iPS cells thus provide an invaluable resource of cell types for modelling diseases, drug or toxicology screening, and patient-specific cell therapy. Significant challenges remain to be overcome before the full potential of human iPS cell technology can be realised. The utilization and practical application of ESC in cell replacement therapy are still in a preliminary stage and need more investigation and clinical trials before they can be accepted as ideal for the treatment of neuromuscular diseases. Nevertheless, the daily increase in experimental findings is reinforcing the hope that ESC will be a versatile source of renewable cells for application in cell replacement therapy (Brignier & Gewirtz, 2010). Therefore, there is enough optimism among the scientists that ESC-based therapies may offer reliable and cost-effective therapeutic substitute for treatment of severe degenerative disorders in the near future. Major objection to hES cell research is focused one ethical reasons. The core reason for objection to hES cell research is that it destroys human blastocysts or embryos, which means it destroys human lives and eventually violates human dignity because of the blastosysts have the same moral value as that of human beings or at least that blastocysts have the potential to develop into human beings (Jung, 2009). Accordingly, research with hESCs is increasing exponentially worldwide, particularly in the United States, where important limitations on research with such cells were overturned in 2009. Furthermore, the US Food and Drug Administration trial using hESC-based therapy in patients with spinal cord injury is now on-going. Nonetheless, a number of substantive scientific and ethical issues remain to be resolved before hESCs can enter the therapeutic mainstream. In the meantime, recent breakthroughs in generating iPSCs would obviate the need to solve the most vexing of these problems. In fact, it seems reasonable to hope that in the next few years many of the enabling issues relevant to iPSCs will be solved, allowing the field of regenerative medicine to deliver on its vast potential promise. Although it is difficult to predict the ultimate utility of stem cell–based therapy at this time, it is not difficult to conclude that this is an extremely important area of scientific research. Open discussions between political bodies and the various interest groups in the scientific, medical, and religious communities need to take place to address the concerns of each and to provide an ultimate solution that is clearly in the interest of humanity.
European Medicines Agency (EMEA) issued the guideline to replace the Points to Consider on the Manufacture and Quality Control of Human Somatic Cell Therapy Medicinal Products (CPMP/BWP/41450/98). In general, when a cell-based medicinal product (CBMP) enters the clinical development phase, the same requirements as for other medicinal products apply. The clinical development plan should include pharmacodynamic studies, pharmacokinetic studies, mechanism of action studies, dose finding studies and randomised clinical trials in accordance to the Directive 2001/20/EC and to the existing general guidances and specific guidances for the condition evaluated. It takes into account the current legislation and the heterogeneity of human cell-based products, including combination products. A risk analysis approach can be used by the applicants to justify the development and evaluation plans and can be a basis for the preparation of a risk management plan. Special problems might be associated with the clinical development of human cell-based medicinal products. Guidance is therefore provided on the conduct of pharmacodynamic/pharmacokinetic studies, dose finding and clinical efficacy and safety studies. The guideline describes the special consideration that should be given to pharmacovigilance aspects and the risk management plan for these products. The active substance of a CBMP is composed of the engineered (manipulated) cells and/or tissues. When the cells in the active substance are genetically modified, the “Note for Guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products” should be followed, which gives details on the quality control, characterisation and preclinical testing of gene transfer vectors. Cell populations which are transformed should be assayed for appropriate and reproducible expression of the newly acquired characteristics. Special attention should be paid to the level and length of expression and quality of the gene product(s) produced by the cells. As far as applicable and practicable, the new characteristics of the cells should be quantified and controlled. During in vitro cell culture, consideration should be given to ensure acceptable growth and manipulation of the isolated cells. The processing steps should be properly designed to preserve the integrity and control the function of the cells and their manipulation should be documented in detail and closely monitored according to specific process controls. Moreover, the duration of cell culture and maximum number of cell passages should be clearly specified and validated. The relevant genotypic and phenotypic characteristics of the primary cell cultures, of the established cell lines and the derived cell clones should be defined and their stability with respect to culture longevity determined. Consistency/repeatability of the cell culture process should be demonstrated and the culture conditions including the media and the duration should be optimised with respect to the intended clinical function of the cells. If genetically modified cells are used in the product, any additional proteins expressed from the vector, such as antibiotic resistance factors, selection markers, should be analysed to determine their presence in the product. Microassay for gene expression profile, flow cytometry and other different techniques allowed these expression studies. CBMP might require administration through specific surgical procedures, method of administration or the presence of concomitant treatments to obtain the intended therapeutic effect. The biological effects of CBMP are highly dependent on the in vivo environment, and may be influenced by the replacement process or the immune reaction either from the patient or from the cell-based product. These requirements coming from the clinical development should be taken into account for the final use of these products. Their standardisation and optimisation should be an integral part of the clinical development studies. Ahead of these considerations it’s not still provided a reproducible method to isolate ESCs even if an ES cell–based therapy would have many advantages: it could allow the transplantation of a more primitive cell with greater replicative potential and patient-specific ES cells could be induced from adult somatic cells. The development of several ESC-based technologies, such as genetic manipulation tools and their potential applications, could accelerate the use of these cells into clinical therapy, even if ethical, logistics and economics concerns need attention in case of ESC-based techniques. There are several major challenges to overcome before iPS cell technology is applied in clinical practice. First, current iPS cells are not “clinical grade”. Genome-integrating viral vectors used for reprogramming are known oncogenes, particularly c-Myc, Oct4 and Klf4, such that iPS cells thus generated are unlikely to be safe for clinical application. Nonetheless recent technological advances, including reprogramming without viral integration such as plasmids or direct reprogramming protein delivery assays could solve this problem (Kiskinis and Eggan, 2010; Saha and Jaenisch, 2009). Despite the challenges in the therapeutic use of iPS cells, preclinical studies provided the proof-of-concept that patient-specific iPS cells can provide an unlimited cell source to produce massive therapeutic cell types, such as cardiomyocytes and MSCs, and can be prepared in an “off the- shelf ” format for cell transplantation. However given the many potential risks of applying autologous iPS cell treatment to human subjects, iPS cell therapies may encounter strict regulatory restrictions.
At now in our opinion the most promising results in the treatment of neuromuscular disorders were obtained using adult stem cells because of many questions are needed to be answer regarding the ES and iPS cells. According to the results described, the most promising possibility for the therapy of muscular dystrophies is a combination of different approaches to obtain the beneficial impact of multiple strategies combined into a single approach, such as cellular therapy associated with gene therapy or pharmacological treatments. One of the most used approach is called autologous transfer in which patient’s own cells are genetically corrected in vitro with lentiviral vectors and then re-implanted to allow the re-expression of functional dystrophin protein. The ‘exon skipping’ approach is an alternative strategy for gene therapy and it is done through AONs that hybridize with the donor and/or acceptor sites of the mutated exon, causing its exclusion from the intact transcript. On the other side, the allogenic transfer implies that the cells isolated from an individual with functional dystrophin will be injected into the patient, allowing problems due to immunorejections or administration of specific immunosuppressive drugs. Several problems arose quickly, such as the low efficiency with which stem cells enter muscle via vasculature, the potential to enhance proliferation of stem cells in culture, the time required in culture for autologous cells prior to implantation back into the patient, the longevity of the transplanted muscle nuclei in vivo, the development of tumours as a consequence of hazardous integration of the provirus. In conclusion the success of the clinical application of adult or embryonic stem cells will be employed to a large-scale production of desired cell type with appropriate functionality, an optimal number of cells for transplant, a modification of less invasive delivery systems and a techniques to label cells for transplant and subsequent tracking of cell fate.
This work has been supported by the Association Monégasque contre les Myopathies (AMM), Telethon grant E36840, Optistem European Project n39\'00i8, the Duchenne Parent Project de France (DPP France), the Associazione La Nostra Famiglia Fondo DMD Gli Amici di Emanuele, Fondazione Cariplo, Fondazione Telethon and the Associazione Amici del Centro Dino Ferrari.
Giant cell arteritis (GCA) is a primary (non-necrotizing granulomatous) vasculitis of autoimmune etiology, which especially affects extra cranial medium-sized arteries (branches of the external carotid arteries-ECAs-particularly the superficial temporal arteries-TAs) and sometimes large-sized arteries (aorta and its major branches). It is also recognized as Horton, temporal, or granulomatous arteritis. It causes narrowing of the artery, leading (by wall thickening) to partial (stenosis) or complete obstruction (occlusion) of local arterial blood flow, its clinical manifestations being expressed by signs of local ischemia [1, 2, 3, 4, 5, 6].
\nGCA is the most common form of vasculitis that occurs in adults and in the elderly, being diagnosed over the age of 50’s. Women are two to three times more affected than men. It is well known that the disease can occur in every racial group but is most common in Caucasians, especially people of northern European descent, and others in northern latitudes. [1, 2, 3, 4, 5, 6].
\nAccording to Hunder [7], and Jennette [8] a complete diagnosis of GCA requires the presence of American College of Rheumatology (ACR) classification modified criteria:
age over 50 years at the onset of the disease;
moderate, bitemporal, recently installed headache;
scalp tenderness, abnormal temporal arteries on inspection and palpation (Figure 1), reduced pulse, jaw claudication (pain in the jaw while/after chewing);
blurred vision or permanent visual loss in one or both eyes (since permanent visual loss due to ischemia is frequent, GCA should be considered an ophthalmic emergency requiring immediate management);
systemic symptoms (fatigue, weight loss, fever, pain in the shoulders and hips: polymyalgia rheumatica);
increased inflammatory markers (erythrocyte sedimentation rate greater than 50 mm/h, C reactive protein greater than 1,5 mg/dl);
representative histologic findings in temporal artery biopsy (TAB): mononuclear cell infiltration or granulomatous inflammation of the vessel wall, usually accompanied with multinucleated giant cells (Figure 2).
Giant cell arteritis (GCA) of the left superficial temporal artery (TA) shows a prominent, tender and nodular artery, that is also hypo pulsating on palpation [9].
The histopathological examination of the left superficial temporal artery biopsy (TAB) noted [10]. (A) Thickened vascular wall with inflammatory infiltration of multinucleated giant cells, (B) epithelioid cells and (C) dissolution of the internal elastic lamina (H&E stain).
Several imaging techniques may be suitable in the diagnosis of GCA. [9] Compared to other imaging techniques, US is considered to be the most suitable in the evaluation of GCA patients, therefor it can easily be performed by the clinician (immediately after the general examination of patient), and it is significantly shortening the waiting period until another investigation is performed. [9, 10, 11, 12, 13, 14, 15, 16].
\nUltrasonography (US) is a safe, noninvasive, without radiations, widespread accessible, fast, and low-cost bedside screening technique which has the unique capacity of studying real-time hemodynamics. It presents the ability to evaluate the anatomy of vessel’s wall, identifying equally parietal abnormalities (wall thickening, hypoechoic plaques, clotting, parietal hematoma, dissections) and the external diameter of the artery; it can rule out both stenosis and occlusion. Therefore, the use of US is widespread in neurological clinical practice, mainly in the evaluation of arterial atherosclerotic process but also for monitoring other diseases such as medium/large-vessel vasculitis. [17, 18, 19].
\nOlah noted that for US imaging of extracranial vessels different modes are being used:
\nB-mode (brightness mode)\n
The strength of the echo is recorded as a bright dot, while the location of different gray dots corresponds to the depth of the target. [17]
\nb. The duplex image\n
It associates a B-mode gray-scale image with pulse-wave (PW) Doppler flow velocities measurements.
The B-mode image represents the anatomical localization of the vessels, indicating the zone of interest where a Doppler sample volume should be placed and where the velocities are measured.
The Doppler angle can be measured correctly when the blood is parallel to the direction of the vessel. [17]
\nc. Color Doppler flow imaging\n
Measure mean frequency shift in each sample volume.
It represents color–coded velocity information, which is superimposed as a color flow map on a B-mode image.
In each sample volume, the color reflects the blood flow velocity in a semi quantitative manner, as well as the flow direction relative to the transducer. Blood flowing toward or away from the transducer is shown by different colors (red and blue). Moreover, fast flow is indicated by a lighter hue and slow flow by a deeper one.
The color flow map indicates the position and orientation of the vessels, as well as the site of turbulent flow or stenosis. Since color flow mapping is based on flow velocity measured by PW technology, aliasing occurs if the frequency shift is higher than half of the pulse repetition frequency (PRF). [17]
\nd. Power Doppler mode\n
Uses the signal intensity of the returning Doppler signal instead of frequency shift.
Power (intensity) of the signal is displayed as a color map superimposed on a B-mode image. Since the Doppler power is determined mainly by the volume rather than the velocity of moving blood, power Doppler imaging is free from aliasing artifacts and much more sensitive to detect flow, especially in the low-flow regions. However, it does not contain information about the flow direction or flow velocity. [17]
The advantages of US over other imaging techniques in GCA are represented by its safety, accessibility, tolerability, fast (may take about 15-20 minutes, if it’s conducted by an experienced sonographer) and the more important, its high resolution (a high –frequency probe offers both an axial and a lateral resolution of 0.1 mm) [19, 20, 21, 22, 23, 24, 25, 26, 27]. The smaller the vessel diameter, the more difficult is to appreciate the vessel wall damages, so that, in this case, the most informative US data are based on Doppler spectral evaluation. This is also valid for the assessment of medium to small vessel inflammation such as intracranial vasculitis. Small vessel vasculitis (the ANCA-associated or the immune complex vasculitis) are not a domain of ultrasound. [19].
\nFurthermore, US has a higher sensitivity than TAB, the last one evaluating only a restricted anatomical region in a systemic disease. Using US, we can reveal pathological characteristics in GCA: non-compressible arteries (compression sign), the wall thickening (“halo” sign), stenosis and vessel occlusion. A normal intima-media complex (IMC) of an artery is represented by US as a homogeneous, hypoechoic or anechoic echo structure delineated by two parallel hyperechoic margins. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nThere is imperative to underline the importance of establishing the arteries that should be routinely examined in a patient suspected for GCA and these are: the TAs, and axillary arteries. If US of these arteries does not reveal suggestive lesions, in the presence of a clear patient history and of an obvious clinical examination, other arteries should be examined: other branches of the ECAs (the internal maxillary, the facial, the lingual, the occipital arteries), the vertebral, the subclavian, the common carotid arteries-CCAs, and the internal carotid arteries-ICAs. [9, 19, 21].
\nRegarding the adequate US equipment for the diagnosis of GCA, modern high-resolution linear probes providing Doppler mode should be used, especially for examination of TAs. We should take into consideration that tissue penetration increases with lower frequencies and the resolution of US increases with higher frequencies. Probes that provide frequencies >20 MHz allow the clearly visualization of the normal IMC of TAs probes with frequencies ≥15 MHz are usually used for detection of minor wall thickening. [19, 21].
\nIn 2012, during the Chapel Hill Consensus Conference [19, 28], large vascular vasculitis (LVV) was well-defined as a vasculitis involving the aorta and its major branches, although any size of artery may be affected. This definition does not state that LVV mainly affects large vessels because in many patients, the number of medium and small arteries affected is greater than the number of large arteries involvement. For example, in GCA, only few branches of the ECAs may be affected when there is involvement of numerous small branches extending into the eye and orbit (e.g., central retinal artery, posterior ciliary arteries). [29, 30] Less frequently, the CCA and the ICA are also affected (Figures 3 and 4). [9].
\nLarge vessels GCA; CT-angiography- occlusion of the left CCA, ECA, and ICA [9].
Large vessels GCA, color Doppler ultrasound in transverse view of the right CCA. Hypoechoic wall swelling with right CCA occlusion [9].
As Sturzenegger pointed up, angiography is not able to illustrate the vessel wall, so as to diagnose the inflammation of the large cervical and cervico-brachial vessels (aorta and its supra-aortic branches), the US can be very useful, since it can define alterations of the vessel wall with the use of B-mode imaging, while Doppler spectral flow velocity evaluation can help identify the stenosis or occlusion of the vessel. [19].
\nColor Doppler Duplex sonography (CDDS) is an excellent device used in screening the large vessels involvement. Agreeing with different authors, including Sturzenegger, there are two ultra-sonographic hallmarks of large vessels GCA:
Vessel wall thickening, that typically is homogeneous, circumferential and over long segments (Figures 4 and 5);
Stenosis, typically revealing slickly tapered luminal tightening (hour glass like) [19, 20, 21, 22, 23, 24, 25, 26, 27]
Large vessel GCA, color Doppler ultrasound in longitudinal view of the right CCA with hypoechoic wall swelling [4].
Remarkably in some cases [9], the common carotid and the internal carotid arteries are also involved (large-vessel GCA) (Figures 3, 4, and 5).
\nExtracranial Duplex sonography investigates almost completely the whole length of the common superficial TAs, including the frontal and parietal branches, and founds that inflammation is segmental (intermittent arterial involvement) [19, 20, 21, 22, 23, 24, 25, 26, 27]. The common superficial TA derives from the ECA. It divides into the frontal and parietal ramus in front of the ear. The distal common superficial TA and the rami are localized between the two layers of the temporal fascia, which is like a bright band at ultrasound examination. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nHigh-resolution color Doppler US can illustrate the vessel wall and the lumen of the TAs. One should use linear probes with a minimum gray scale frequency of 8 Mhz. Color frequency should be about 10 Mhz. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nThe pulse repetition frequency (PRF) should be about 2.5 kHz as maximum systolic velocities are rather high (20-100 cm/s). Steering of the color box and the Doppler beam should be maximal as the rami are parallel to the probe. It is important that the color covers the artery lumen exactly. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nThe sonographer should perform at least 50 Duplex ultrasound of the TAs of subjects without GCA to be sure about the appearance of normal TAs before starting to evaluate patients with GCA. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nThe investigation should begin with the TA, using the longitudinal scan. The probe should then be moved along the course of the TA to the parietal ramus. On the way back one should delineate the TA in transverse scans. Using the transverse scan, one can find the frontal ramus, which should then be delineated in both scans (longitudinal and transverse). If the color signal indicates localized aliasing and diastolic flow, one should use the pw-Doppler mode to confirm the presence of stenosis. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nIn 1997 Schmidt et al. proved that the most specific (almost 100% specificity) and sensitive (73% sensitivity) sign for GCA was a concentric hypo-echogenic mural thickening, dubbed “halo”, which the authors interpreted as “vessel wall edema”. [24].
\nOther positive findings for GCA are the presence of occlusion and stenosis. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nIn conclusion, there are three important items in the ultrasound diagnosis of temporal arteritis:
“dark halo” sign – a typically homogeneous, hypoechoic, circumferential wall thickening around the lumen of an inflamed TA - which represents vessel wall edema and a characteristic finding in temporal arteritis/GCA. It is well delineated toward the lumen (Figure 6).
stenosis are documented by blood-flow velocities, which are more than twice the rate recorded in the area of stenosis compared with the area before the stenosis, with wave forms demonstrating turbulence and reduced velocities behind the area of stenosis (Figure 7).
acute occlusions, in which the US image is comparable to that of acute embolism in other vessels, showing hypoechoic material in the former artery lumen with absence of color signals. [19, 20, 21, 22, 23, 24, 25, 26, 27]
Color Doppler ultrasonography (CDUS) of the right TA shows a hypoechoic halo around the lumen in transverse view (arrow). The “halo sign” corresponds to edema of the artery wall. [11].
Longitudinal view of the right TA by color Doppler ultrasonography (CDUS) shows a hypoechoic halo of the TA and the presence of turbulent and weak flow, suggesting the presence of stenosis. The PSV is 1 m/s, that is double compared to the segment without stenosis. [11].
Related ultrasound patterns can be found in other arteries: the facial, the internal maxillary, the lingual, the occipital, the distal subclavian and the axillary arteries.
\nThe best time to perform ultrasound investigation is before initiating the corticosteroid treatment, or in the first 7 days of treatment, since with corticosteroid therapy the” halo” revealed by TAs ultrasound disappears within 2-3 weeks. The wall inflammation, stenosis, or occlusions of the larger arteries (CCA, ICA) remain for months, despite corticosteroid treatment. However, the diagnosis process should not postpone the initiation of therapy. Ultrasound may also detect inflamed TAs in patients with clinically normal TAs. Some patients with the clinical image of polymyalgia rheumatica, but with hidden TAs may be diagnosed using ultrasonography. [9, 10, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 25, 26, 27].
\nIn 2010, Arida et al. [26] evaluated a number of studies that examined the sensitivity and specificity of the “halo” sign confirmed by TA ultrasound (US) for GCA diagnosis versus the American College of Rheumatology (ACR) 1990 criteria for the classification of this vasculitis (used as a reference standard). Only 8 studies involving 575 patients, 204 of whom received the final diagnosis of GCA, achieved the technical quality criteria for US. This meta-analysis disclosed a sensitivity of 68% and a specificity of 91% for the unilateral “halo” sign, as well as 43% and 100%, respectively, for the bilateral “halo” sign in TA US for GCA diagnosis when the 1990 ACR criteria are used as the reference standard. The authors established that the halo sign in US is of great utility in diagnosing GCA. [19, 20, 21, 22, 23, 24, 25, 26, 27].
\nIn the case of consistent clinical and sonographic results, temporal arteries biopsy (TAB) does not appear to be useful and justified. [19, 27].
\nSturzenegger affirmed that differential diagnosis with arteriosclerosis is important in patients over 50 years, taking into consideration that GCA with large vessels disease disturbs almost exclusively this category of patients. There are some characteristic features of the arteriosclerotic wall: the thickening usually appears less homogeneous; there are calcified arteriosclerotic plaques ulcers; stenosis extends over shorter segments, they are not concentric, not tapering, and location of lesions differs (e.g., mainly bifurcations). [19].
\nBesides, agreeing to Sturzenegger, differential diagnosis with the other LVV, especially Takayasu arteritis, has to be reflected:
Takayasu arteritis usually affects women below the age of 40 years;
symptoms like tender scalp or polymyalgia syndrome are exceptional;
the involvement of CCA is more frequent in Takayasu arteritis, while the involvement of temporal artery in Takayasu arteritis is not known;
US image of wall thickening (“halo”) is brighter in TA than in GCA probably due to a larger mural edema in GCA which is a more acute disease than TA. Reflected. [19, 20, 21, 22, 23, 24, 25, 26, 27]
Approximately 25% of patients with temporal artery biopsy (TAB) - proven GCA have ophthalmologic complications: usually unilateral visual loss (due to the vasculitic involvement of orbital vessels:
of posterior ciliary arteries (PCAs) - represented by arteritic anterior ischemic optic neuropathies (A-AION), or
of central retinal artery (CRA) - represented by central retinal artery occlusion (CRAO). [31, 32, 33, 34, 35]
Schmidt compared the results of TAs-US examinations with the occurrence of visual ischemic complications (A-AION, CRAO, branch retinal artery occlusion, diplopia, or amaurosis fugax) in 222 consecutive patients with newly diagnosed, active GCA. [21, 22, 23, 24].
\nHowever, findings of TAs US did not correlate with eye complications. [21, 22, 23, 24].
\nThis is the reason why we always have to exam the orbital (retrobulbar) vessels in GCA patients or in patients with unilateral abrupt visual loss [9, 10, 11, 12, 13, 14, 15, 16] (Figure 8A,B).
\nColor Doppler imaging (CDI) of orbital (retro-bulbar) vessels: (A). central retinal artery (CRA); (B). posterior ciliary arteries (PCAs) [15].
The ophthalmic artery (OA) branches in several arteries, including (Table 1):
the central retinal artery (CRA) (Figure 8 A), and
the posterior ciliary arteries (nasal and temporal branches-nPCAs, tPCAs) [28, 31, 32] (Figure 8B), (Table 1). [15, 28, 31, 32]
Parameter | \nOA | \nCRA | \nPCA (temporal) | \nPCA (nasal) | \nSOV (superior ophthalmic vein) | \n
---|---|---|---|---|---|
PSV (cm/s) | \n45,3 ± 10,5 | \n17,3 ± 2,6 | \n13,3 ± 3,5 | \n12,4 ± 3,4 | \n10,2 ± 3,8 | \n
EDV (cm/s) | \n11,8 ± 4,3 | \n6,2 ± 2,7 | \n6,4 ± 1,5 | \n5,8 ± 2,5 | \n4,3 ± 2,4 | \n
RI | \n0,74 ± 0,07 | \n0,63 ± 0,09 | \n0,52 ± 0,10 | \n0,53 ± 0,08 | \n\n |
OA finishes in the a. supra-trohlearis and A. dorsalis nasi.
\nStandard neurovascular ultrasound machines equipped with linear-array transducers emitting 6-12 MHz (up to 15 MHz) are adequate for identifying (by Color Doppler sonography), and measuring (by spectral analysis pulsed Doppler sonography) the blood flow in the orbital vessels: the OA, the CRA and central retinal vein (CRV), PCAs, and the superior ophthalmic vein (SOV). [28, 31, 32].
\nThe CRA, a distal branch of the OA, enters the optic nerve (ON) approximately 1-1.5 cm distal from the bulbus coming from the dorsolateral direction. Parallel to this is the CRV.
\nThe PCAs are located near the optic nerve (ON) (the nasal-nPCA and the temporal-tPCA branches). [28, 31, 32].
\nIf the vessels are difficult to display, the power should be elevated for a short time if the clinical question is important. [28, 31, 32].
\nThe optic nerve head (ONH) consists of (from anterior to posterior):
the surface nerve fiber layer - mostly supplied by the retinal arterioles. The cilioretinal artery, when present, usually supplies the corresponding sector of the surface layer. [36, 37, 38, 39, 40]
the prelaminar region - situated anterior of the lamina cribrosa. It is supplied by centripetal branches from the peripapillary choroid. [36, 37, 38, 39, 40]
the lamina cribrosa region - supplied by centripetal branches from the posterior ciliary arteries (PCAs), either directly or by the so-called arterial circle of Zinn and Haller (when is present). [36, 37, 38, 39, 40]
the retrolaminar region - is the part of the ONH that lies immediately behind the lamina cribrosa. It is supplied by two vascular systems: the peripheral centripetal and the axial centrifugal systems. The previous represents the main source of stream for this part. It is formed by recurrent pial branches arising from the peripapillary choroid and the circle of Zinn and Haller (when present, or the PCAs instead). In addition, pial branches from the central retinal artery (CRA) also supply this part. The latter is not present in all eyes. When present, it is formed by inconstant branches arising from the intraneural part of the CRA.
From the description of the arterial supply of the ONH given above, it is obvious that the PCAs are the main source of blood supply to the ONH. [36, 37, 38, 39, 40].
\nThe blood flow in the ONH depends upon [36, 37, 38, 39, 40]:
resistance to blood flow - depends upon the condition and caliber of the vessels supplying the ONH, which in turn are influenced by: the efficiency of auto-regulation of the ONH blood flow, the vascular variations in the arteries feeding the ONH circulation, and the rheological properties of the blood.
arterial blood pressure (BP) - both arterial hypertension and hypotension can influence the ONH blood flow in several ways. In an ONH, a fall of blood pressure below a critical level of auto-regulation would decrease its blood flow. Decrease of BP in the ONH may be due to systemic (nocturnal arterial hypotension during sleep, intensive antihypertensive medication, etc.) or local hypotension.
intra-ocular pressure (IOP) - there is an opposite relationship between intra-ocular pressure and perfusion pressure in the ONH.
The blood flow in the ONH is calculated by using the following formula:
\nPerfusion pressure = Mean BP minus intraocular pressure (IOP).
\nMean BP = Diastolic BP + 1/3 (systolic - diastolic BP) [6, 13].
\nAION is the consequence of an acute ischemic disorder (a segmental infarction) of the ONH supplied by the PCAs. Blood supply interruption can occur with or without arterial inflammation. Therefore, AION is of two types: non-arteritic AION (NA-AION) and arteritic AION (A-AION). The prior is far more common than the last, and they are distinct entities etiologically, pathogenically, clinically and from the management point of view. [36, 37, 38, 39, 40].
\nA history of amaurosis fugax before an abrupt, painless, and severe loss of vision of the involved eye, with concomitant diffuse pale optic disc edema is extremely suggestive of A-AION. None of these symptoms are found in NA-AION patients. [36, 37, 38, 39, 40].
\nIn acute stage, blood flow cannot be detected in the PCAs in the clinically affected eye of any of the GCA patients with A-AION. Low end diastolic velocities (EDV) and high resistance index (RI) are identified in all other orbital vessels (including the PCAs in the opposite eye) of all A-AION patients. [9, 10, 11, 12, 13, 14, 41].
\nOver 7 days, Spectral Doppler analysis of the orbital vessels highlights blood flow alterations in all A-AION patients even with a high-dose corticosteroids therapy. Severely reduced blood flow velocities (especially EDV) in the PCAs of the affected eye (both nasal and temporal branches), compared to the unaffected eye, are observed. An increased RI in the PCAs is noted (the RI is higher on the clinically affected eye as compared to the unaffected eye). [9, 10, 11, 12, 13, 14, 41] (Figure 9A,B).
\nCDI of the PCAs in A-AION: (A). Decreased EDV in the nasal PCAs of the clinically affected right eye, and (B) of the clinically unaffected left eye.
Fewer abnormalities are detected in the CRAs: high RI are measured in both sides, with decreased peak systolic velocities (PSV) in the CRA of the clinically affected eye. [9, 10, 11, 12, 13, 14, 41].
\nSimilar abnormalities are noted in the OAs: high RI are measured in both sides. [9, 10, 11, 12, 13, 14, 41].
\nAt 1 month, after treatment with high-dose corticosteroids, CDI examinations of orbital blood vessels reveals that blood flow normalization is slow in all A-AION patients. [9, 10, 11, 12, 13, 14, 41].
\nIn conclusion, the Spectral Doppler Analysis of the orbital vessels in A-AION indicates (after several days of corticosteroid treatment) low blood velocities, especially EDV, and high RI in all orbital vessels, in both orbits. These signs represent characteristic signs of the CDI of the orbital vessels in A-AION. [9, 10, 11, 12, 13, 14, 41].
\nIn contrast, the patients with NA-AION present the following characteristics in acute stage, and at 1 week of evolution:
minor reduction of PSV in PCAs (nasal and temporal) in the affected eye, compared to the unaffected eye.
slight decrease of PSV in CRA of the affected eye, due to papillary edema. [9, 10, 11, 12, 13, 14, 41]:
in OAs, PSV are variable: normal to decreased, according to ipsilateral ICAs status.
Severe ICA stenosis (≥70% of vessel diameter) combined with an insufficient Willis polygon led to diminish PSV in ipsilateral OA. [9, 10, 11, 12, 13, 14, 41].
\nIn 1 month, CDI examinations of orbital blood vessels reveal that blood flow normalization is reached. The exceptions are the cases with severe ipsilateral ICA stenosis/occlusion. [9, 10, 11, 12, 13, 14, 41].
\nIn conclusion, in NA-AION, blood velocities and RI in PCAs are preserved. Similar results were obtained in other studies. [9, 10, 11, 12, 13, 14, 41].
\nFluorescein angiogram and CDI of the orbital vessels data support the histopathological evidence of involvement of the entire trunk of the PCAs in the A-AION (impaired optic disc and choroidal perfusion in the patients with A-AION). On the other hand, in the NA-AION, the impaired flow to the optic nerve head (ONH) is distal to the PCAs themselves, possibly at the level of the para-optic branches (only 1/3 of the flow of the PCAs). [36, 37, 38, 39, 40].
\nThese branches supply the ONH directly (impaired optic disc perfusion, with relatively conservation of the choroidal perfusion). [36, 37, 38, 39, 40].
\nExtremely delayed or absent filling of the choroid has been depicted as a fluorescein angiogram characteristic of arteritic AION and has been suggested as one useful factor by which A-AION can be differentiated from NA-AION. [36, 37, 38, 39, 40].
\nCRAO is the result of an abrupt diminuation of blood flow in CRA, severe enough to cause ischemia of the inner retina. Due to the fact that there are no functional anastomoses between choroidal (PCAs) and retinal circulation (CRA), CRAO determines severe and permanent loss of vision. Therefore, it is very important to identify the cause of CRAO, in order to protect the contralateral eye. Frequently, the site of the blockage is within the optic nerve substance and for this reason, it is generally not visible on the ophthalmoscopy. The majority of CRAO are caused by thrombus formation due to systemic diseases, including GCA. For this reason, all patients with CRAO should undergo a systemic evaluation. [42, 43, 44].
\nThe patients with an unilateral CRAO present at the Spectral Doppler analysis of the retrobulbar vessels the following aspects [9, 15, 16]:
an elevated RI in the CRAs (the RI is higher on the affected side, than it is on the unaffected side); with severe diminished blood flow velocities (especially EDV) in the CRA.
fewer abnormalities are observed in the PCAs, and in the OAs (Figure 10).
CDI of orbital vessels revealed severely diminished EDV and high RI in both CRAs (a, b) despite the fact that the left eye had the normal aspect at ophthalmoscopy. Fewer abnormalities were observed in the PCAs (c, d). [15].
Other imaging techniques, such as high-resolution magnetic resonance imaging (MRI), magnetic resonance-angiography (MR-A), computer tomography angiography (CT-A), positron emission tomography (PET) provide valuable information regarding the structure of large vessels, highlighting with much greater precision the thoracic aorta, compared with US. [45, 46, 47].
\nThere are few studies that compared US with other imaging techniques. Some of them indicated that there is a good correlation between US and PET, even though PET might have a little more sensitivity for vertebral arteries examination. [45, 46] 18F-fluorodeoxyglucose-positron emission tomography/ computed tomography (FDG-PET/CT) has a higher sensitivity for detection of large arteries and aortic involvement - analysis of the arterial wall. [45, 46] The diagnostic power of high-resolution MRI and color-coded duplex US of extra-cranial arteries in detecting GCA are equivalent [47].
\nThe disadvantages of this techniques are: they are more expensive, hardly accessible, some of them are limited by invasiveness, nephrotoxicity (angiography) and exposure to high radiations (CT,PET), this is why they might be unnecessary (excepting those patients with exclusively thoracic aorta involvement) and are not accepted as diagnostic methods in GCA. They should only be used when interventions are required [45, 46, 47].
\nAll these imaging techniques should always be performed by well-trained specialists, using suitable equipment and operational protocols. [45, 46, 47].
\nNevertheless, US is particularly useful in examining the orbital vessels. [9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 41].
\nThe diagnostic work-up of AION benefits from the combined used of fluorescein angiography and noninvasive multimodal imaging, including CDI of the orbital vessels and structural Optical Coherence Tomography (OCT) of the optic nerve head (ONH) and OCT angiography [10, 48]. They provide very detailed information regarding the structural (retinal nerve fiber layer-RNFL-thickness/optic disc edema) and vascular impairments (microvascular defects-vessel tortuosity, and vessel density reduction) of the ONH, respectively [10, 48].
\nUS should be used as a first-line diagnostic investigation for patients presenting with clinical and biological features suggestive for GCA, taking into consideration that it has a high sensitivity to detect vessel wall thickening (dark hallo sign) in the case of large/medium vessels. In a few cases of our studies, the CCAs and the ICAs were also involved.
\nIn consequence, in our department, CCDS has emerged as a safe and reliable alternative to TAB as a point of care diagnostic tool in the management of temporal arteritis.
\nThe eye involvement in GCA is frequent and consists in A-AIONs or CRAO, with abrupt, painless, and severe loss of vision of the involved eye.
\nBecause findings of TAs US do not correlate with eye complications in GCA, CDI of the orbital vessels is of critical importance, in order to quickly differentiate the mechanism of eye involvement (arteritic, versus non-arteritic). This US tehnique may be helpful to detect the blood flow in the orbital vessels, especially in cases of opacity of the medium, or when the clinical appearance of ophthalmologic complications in temporal arteritis is athypical.
\nThe Spectral Doppler Analysis of the orbital vessels in GCA with eye involvement reveals low blood velocities, especially EDV, and high RI in all orbital vessels, in both orbits, for all patients (especially on the affected side).
\nA huge advantage of CDI of orbital vessels is that it provides immediate information that can be used to inform treatment decisions, including a potential reduction in loss of sight and avoidance of unnecessary long-term steroid treatment by early exclusion of mimics.
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