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Research Report
Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida George M. Smitha,b, Barbara Krynskaa,c,n a
Shriners Hospitals Pediatric Research Center and Center for Neural Repair and Rehabilitation, Temple University School of Medicine, Philadelphia, PA 19140, USA b Department of Neuroscience, Temple University School of Medicine Philadelphia, PA 19140, USA c Department of Neurology, Temple University School of Medicine Philadelphia, PA 19140, USA
art i cle i nfo
ab st rac t
Article history:
Myelomeningocele (MMC) is a devastating spinal cord birth defect, which results in
Accepted 25 November 2014
significant life-long disabilities, impaired quality of life, and difficult medical management. The pathological progression of MMC involves failure in neural tube and vertebral arch
Keywords:
closure at early gestational ages, followed by subsequent impairment in spinal cord and
Myelomeningocele
vertebral growth during fetal development. MMC is irreversible at term. Thus, prenatal
Spina bifida
therapeutic strategies that interrupt progressive pathological processes offer an appealing
Birth defects
approach for treatment of MMC. However, a thorough understanding of pathological
Spinal cord injury
progression of MMC is mandatory for appropriate treatment to be rendered.
Vertebral defects
This article is part of a Special Issue entitled SI: Spinal cord injury.
Micro-computed tomography
1.
Introduction
In this review, we discuss the MMC spinal cord lesion with emphasis on the associated vertebral defects and how the use of sensitive and quantitative outcome measures can precisely identify and quantify changes in spinal cord and vertebral defects to better characterize progression and identify treatments for MMC. The accurate identification and quantitative evaluation of MMC abnormalities is challenging because MMC is a highly complex defect, characterized by a dynamic malformation process during gestation and varying degrees of neural tissue loss and vertebral defects. Recent refinements
& 2014 Elsevier B.V. All rights reserved.
in available imaging techniques can be used to precisely identify and quantitatively evaluate the defect or repair status of MMC. This review highlights the background and rationale for the use of 3D high resolution micro-computed tomography (micro-CT) imaging and digital analyses methods for improved quantitative assessment of MMC defects in animal models. We propose that the inclusion of 3D imaging and quantitative measures of MMC defects using high-resolution micro-CT will provide a deeper understanding of pathophysiology or repair strategy of MMC in animal models, and make the studies of MMC more effective and efficient.
n Correspondence to: Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3500 N Broad Street, Philadelphia, PA 19140, USA. Fax: þ1 215 707 8235. E-mail address: [email protected] (B. Krynska).
http://dx.doi.org/10.1016/j.brainres.2014.11.053 0006-8993/& 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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2.
MMC the problem
Myelomeningocele (MMC), the most severe form of spina bifida, is a common and devastating congenital malformation and a major source of disabilities and morbidity in children. MMC affects less than one of 1000 live births in the United States (Parker et al., 2010). About 75% of affected children survive to adulthood, but, live with significant life-long physical disabilities (Bowman et al., 2001). The exact cause of spina bifida is unknown; however, teratogenic, nutritional and genetic factors are associated with the development of spina bifida (Alles and Sulik, 1990; Kibar et al., 2007; Ornoy, 2009). Although certain risk factors are known to be associated with the development of spina bifida, and the prevalence of spina bifida has declined in the post-fortification era with the use of folic acid supplementation to reproductive age women, spina bifida still can affect the pregnancy of any women, at any age, or of any baseline health status (Moldenhauer, 2014; Osterhues et al., 2013). Despite the clinical impact of MMC, there is no cure for this devastating congenital defect. Children affected by MMC face significant life-long physical disabilities including, leg paralysis and sensory loss, bowel and bladder dysfunctions, skeletal deformations, and the Arnold-Chiari II malformation with secondary hydrocephalus (Dias and McLone, 1993; Hunt, 1990; Hunt and Poulton, 1995). These disabilities can lead to other medical conditions that require clinical care. The most common secondary conditions associated with MMC are urinary tract infections followed by complications from devices/ implants and various types of skin injuries e.g., pressure ulcers resulting from prolonged sitting in one position (Dicianno and Wilson, 2010; Verhoef et al., 2004). Approximately 30–50% of MMC patients rely on a wheelchair for mobility (Hunt and Oakeshott, 2003; McDonnell and McCann, 2000). The individuals living with the defect have a profoundly diminished quality of life and ability to work. They usually require lifelong support and institutional care. To optimize the quality of life and improve the functional outcomes of these individuals, lifelong multidisciplinary care including neurosurgery, urology, orthopedics and rehabilitation is needed (Sandler, 2010). Treatment and management of patients with spina bifida continue to have a huge economic burden on the health care system (Centers for Disease Control and Prevention (CDC) 2007; Radcliff et al., 2012), and its physical and emotional burden on children and their families is devastating.
2.1.
Spinal cord injury in MMC
MMC is a complex congenital defect of neural tube and vertebral arches closure, characterized by a dynamic pathological process during gestation that results in the protrusion of the spinal cord and meninges through a defect in the spine leading to the spinal cord injury at the level of MMC. The condition can result in varying sensory and motor deficits below the affected neurologic level of the lesion, and a variety of associated problems, including hindbrain herniation, hydrocephaly, loss of bowel and bladder control and various orthopedic complications (Dias and McLone, 1993; Hunt 1990; Hunt and Poulton, 1995; Hunt and Oakeshott, 2003; Tomlinson and Sugarman, 1995). A better understanding of spinal cord injury in MMC has been revised
during the last decade, resulting in the “two-hit hypothesis”. The first hit, denotes the initial defect of neurulation and the secondary hit represents the subsequent in utero acquired damage to the openly exposed spinal cord (Meuli and Moehrlen, 2013). Although, failure during neurulation is the initial cause for the spinal cord malformation, compelling clinical as well as experimental evidence suggest that the exposed spinal cord results in secondary in utero damage, predominantly causing the neurological deficit associated with MMC (Heffez et al., 1990; Meuli et al., 1995b; Sival et al., 1997; Stiefel et al., 2007). Human studies show that the openly exposed spinal cord is histologically intact in early gestation, showing little damage; however, progressive damage sometimes leading to complete loss, was observed in late stages of gestation (Hutchins et al., 1996; Meuli et al., 1997). In support of the in utero acquired secondary damage, experimental evidence demonstrated that surgically induced in utero exposure of the spinal cord (human-like spina bifida lesions) in experimental animals (rat, pig, sheep) led to spinal cord damage of the exposed segments and functional loss observed at birth (Heffez et al., 1993; Meuli et al., 1995a; Meuli et al., 1995b). The definitive secondary mechanisms underlying the pathogenesis of spinal cord injury remain unclear. Although, it has been proposed that the in utero acquired damage of the spinal cord includes, mechanical, degenerative, and possibly inflammatory damage to the openly exposed spinal cord tissue caused by exposure to amniotic fluid, traumatic injury within the amniotic space, hydrodynamic pressure, or a combination of these (Heffez et al., 1993; Meuli et al., 1995a; Meuli et al., 1995b; Meuli and Moehrlen, 2013). Thus, prenatal therapeutic strategies that interrupt the pathological processes offer an appealing approach for treatment of MMC.
2.2.
Prenatal and postnatal repair for MMC
Despite the clinical impact of MMC, there is no cure for this devastating defect. Prevention strategies can reduce the onset of MMC; however, uncertainty remains regarding its etiology and the underlying mechanisms, which is a prerequisite for effective preventive strategies. Prophylaxis treatment with folic acid has proven efficacious in reducing total numbers, however, it helps only a fraction of pregnant women, and thus a significant portion of neural tube defects still persists (Oakley, 2010; Osterhues et al., 2013; Parker et al., 2010). Once spina bifida occurs folic acid is not helpful. Therefore, women carrying a fetus diagnosed with MMC have few options, including standard surgical closure shortly after birth, closure of the defect during the fetal period in selected cases, or termination of pregnancy. Postnatal surgical closure of MMC defect is associated with poor neurological outcomes because the postnatal therapy cannot prevent or reverse the neurological injury, since the affected spinal cord is destroyed and neurologic function lost before birth (Adzick et al., 2011; Simpson and Greene, 2011). Because experimental and clinical evidence shows progressive damage to the defective area during fetal development in utero, prenatal surgical closure was thought to be a potential treatment (Heffez et al., 1990; Hutchins et al., 1996; Meuli et al., 1995b; Meuli et al., 1997; Sival et al., 1997; Stiefel et al., 2007). Indeed, animal experiments of in utero repair after surgically created defects showed improved neural function (Heffez et al., 1993; Meuli et al., 1996).
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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Given that prenatal diagnosis of neural tube defects and surgical techniques have improved over the past several years, the concept of in utero repair of MMC in humans became feasible. In utero surgical closure of MMC improves the functional neurological outcomes including the ability to walk without orthotics or devices in some patients, a reduction in hindbrain herniation and a decreased need for ventriculoperitoneal shunting in patients who underwent prenatal compared with those who had postnatal closure (Adzick et al., 2011; Simpson and Greene, 2011). Currently, more than ten centers in the United States are performing in utero MMC repair, which now is a clinical option for the management of prenatally diagnosed MMC in eligible patients (Moldenhauer, 2014). However, despite the supporting evidence that the prenatal surgical repair is more successful than postnatal MMC repair, the surgical procedure occur well after the initial exposure of the spinal cord to the amniotic environment, and does not completely repair the spinal cord injury, so significant morbidity remains in most patients. In addition, there are significant maternal and fetal risks associated with prenatal surgery and the impact on the long-term outcomes of children who underwent fetal surgery for MMC is currently unknown and requires further study (Moldenhauer, 2014). Thus, further research of this defect is needed to advance the understanding of MMC development and identify new treatment approaches, through which the concept of preventing or reversing neurological impairments caused by MMC may be achieved.
2.3.
Vertebral (mesenchymal) defects in MMC
Spinal cord injury, which occurs with MMC is thought to be a progressive intrauterine phenomenon, rather than completely a condition induced by defective neurulation. The etiology and pathogenesis of MMC is unclear and most likely mulifactorial. The accepted view has been that the neuroepithelium is initially involved in the failure of neural tube closure and that the associated vertebral (mesenchymal) anomalies are secondary (Copp and Greene, 2013; Finnell et al., 2000). While spina bifida is often synonymous with neural tube defects, other etiologies have also been proposed for spina bifida development. Analysis of mouse models of spina bifida suggests that the mesoderm surrounding the neural tube may be initially involved in development of this defect (Helwig et al., 1995; Payne et al., 1997; Pickett et al., 2008; Takahashi et al., 1992). In experimental systems MMClike defects can be induced by retinoic acid overexposure (Alles and Sulik, 1990; Danzer et al., 2005; Tibbles and Wiley, 1988). Although the exact mechanisms through which alltrans retinoic acid induces MMC remains largely unknown, it has been proposed that excessive cell death of embryonic mesenchymal cells may play a role in the induction of these malformations (Alles and Sulik, 1990). Early studies on human fetuses with MMC suggest that the musculoskeletal system may be initially affected resulting in open exposure of the spinal cord, followed by progressive in utero damage to the spinal cord (Hutchins et al., 1992; Jordan et al., 1991). These findings support the hypothesis that some categories of human MMC may arise from a failure of the vertebrae to close at the midline, suggesting that the primary abnormality may be in the musculoskeletal system. Accordingly, animal
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models of MMC created by fetal surgery provide evidence that open exposure of the normal spinal cord to the intrauterine environment results in MMC-type lesion that have clinical and morphological similarities to human MMC (Heffez et al., 1990; Hutchins et al., 1996; Meuli et al., 1995b). Surgical lesions that remove the vertebral arches without directly damaging the spinal cord resulted in herniation of the spinal cord and its membranes through the vertebral defect, forming a cystic human-like MMC (Meuli et al., 1995b). In addition, earlier studies in primates also show that MMC created by fetal laminectomy resulted in the spinal cord injury and MMClike neurologic deficits at birth (Michejda, 1984). Immediate in utero repair of the bone deficit after fetal laminectomy resulted in these animals having near normal neurological scores at birth (Michejda, 1984). Although, several etiologies have been proposed for spina bifida, all with unknown casual mechanisms, it appears that the sentinel pathology in MMC is in the defect in the mesodermallyderived musculoskeletal tissues. The neurologic injury occurs secondary to these defects. Convincing experimental and clinical evidence indicates no signs of trauma or degeneration of spinal tissue at early gestational time points, and spinal cord degeneration appears to progress in severity with gestational age (Keller-Peck and Mullen, 1996; Meuli et al., 1997; Selcuki et al., 2001; Stiefel et al., 2007). With naturally occurring MMC, part of the spinal cord pushes through congenitally unclosed openings in overlying vertebrae and protrudes from the fetus' back into amniotic space, followed by progressive damage to the spinal cord leading in some cases to complete loss observed in late stages of gestation (Hutchins et al., 1996; Meuli et al., 1997). The extent of neurological deficits and disabilities associated with MMC is dependent on the severity of the defect. Although, the intrauterine conditions are thought to negatively impact spinal cord development leading to functional loss observed at birth, there is little experimental data, e.g., from animal models for the quantitative assessment of MMC pathology. Given the nature of the MMC defect, one potential quantitative means of assessing defect severity during gestation is by measuring impairment of fetal vertebral development with respect to the degree of spinal cord damage, at different gestation periods. In combination with neurological assessment of animals, the information obtained from these studies would establish a correlation between abnormal fetal spine development, the spinal cord injury and loss of neurologic function before birth. A comprehensive quantitative analysis of MMC pathology would improve an assessment of MMC, while measurements at different gestation periods should advance the understanding of the developmental trajectory of MMC.
2.4.
Quantitative assessment of MMC in an animal model
Although several animal models of MMC have been developed to study the pathogenesis and treatment approaches, limited work has been done to improve the quantitative outcome measures of MMC. At present, anatomical MMC assessment in animal models is usually performed by conventional 2D histopathological analysis of spinal cord defect and whole-body skeleton staining (Cai et al., 2007; Danzer et al., 2005). While histological methods offer unique capabilities, these traditional techniques may be inadequate to fully visualize and
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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quantitatively evaluate MMC defects. The methods of wholebody skeleton staining are invasive, destructive techniques that severely limit precise 3D visualization and quantitative assessment of skeletal defects in MMC fetuses and are time consuming. The invasiveness and 2D analyses of histological sections are also the main disadvantages limiting the opportunity for full 3D visualization and quantitative evaluation of fetal spinal cord abnormalities in MMC. In addition, the complete assessment of MMC defects can be difficult to perform in the same animal using traditional techniques. For example, due to the destructive nature of histological techniques, analysis of the vertebral level and skin level of the MMC lesion in mice after whole skeletal preparations was performed using the correlation of the cranial end of the skeletal defect with the suturemarked cranial end of the skin defect (Stiefel and Meuli, 2008). Advanced techniques, e.g. MRI can provide 3D images of MMC in animal model (Danzer et al., 2005) however, data acquisition is usually time-consuming and it is difficult to obtain highresolution imaging in small animals adequate for quantitative analysis of MMC, in particular the skeletal defect. Micro-computed tomography (micro-CT) is a non-invasive whole volume imaging technique that provides high contrast between soft tissue and bone and offers quantitative data on bone morphology and architecture and has been helpful in characterizing developmental defects of bone (Bouxsein et al., 2010; Ford-Hutchinson et al., 2003; Oest et al., 2008; Vasquez et al., 2013). The great detail with which the anatomy can be visualized and the abnormalities quantified (Cunningham and Black, 2009; Oest et al., 2008), makes high resolution micro-CT an attractive imaging technique for assessing the characteristics of MMC skeletal defects in animal models. As an initial step toward the development of improved methods for assessment of MMC, we have recently described the assessment of MMC skeletal defects in an established retinoic acid induced rat model of lumbosacral MMC using high-resolution micro-CT imaging combined with digital quantification methods (Barbe et al., 2014). The retinoic acid induced rat model of MMC is developmentally and phenotypically analogous to human MMC, and is relevant for investigating the pathogenesis and in utero treatments of MMC (Danzer et al., 2005; Danzer et al., 2011; Watanabe et al., 2011; Zhao et al., 2008). Using 3D micro-CT images of rat vertebral defects and corresponding normal vertebra we observed, in detail, skeletal defects in lumbosacral vertebra of MMC rats, including morphological defects of individual dorsal vertebral arches. The defect in lambosacral vertebra appeared in the form of delayed or absent ossification. Butterfly-shaped, dumbbellshaped, non-fused, and absent to nearly absent vertebra centra were identified using high resolution 3D micro-CT images of lumbosacral vertebrae (Barbe et al., 2014). We show highresolution micro-CT to be an especially powerful method of analysis that provides not only anatomical details of MMC defect, but also adequate 2D and 3D images for digital quantification not previously achieved in models of MMC. We were able to nondestructively quantify the distance between the ends of the vertebral arches in the retinoic acid induced rat model of MMC and showed a significant increase in the distance between dorsal vertebral arches from L5 to S4 when compared to the control group. In addition, micro-CT method is a powerful tool for assessing overall skeletal phenotype and it allowed us to visualize supernumerary ribs in fetuses with MMC. As often
reported in the literature, micro-CT is the imaging technique of choice for the measurement and visualization of bone structure. It can also be used for quantitative analysis of various fetal skeletal development parameters including bone mineral density as well as identification of skeletal malformations with high degree of accuracy (Campbell and Sophocleous, 2014; Cunningham and Black, 2009; Oest et al., 2008). Micro CT has also become a popular imaging technique to study soft tissues (Campbell and Sophocleous, 2014). Recent, studies indicate that ex vivo micro-CT imaging, with the aid of staining agents that make soft tissues radiopaque, has the potential for non-invasive imaging and quantitative analysis of soft tissues in 3D including spinal cord (Saito and Murase, 2013; Tahara and Larsson, 2013; Vegesna et al., 2012; Vegesna et al., 2013). Although, the optimization of staining methods is needed to distinguish soft tissue from bone tissue and delineate spinal cord, these studies indicate the potential of micro-CT for evaluation of both vertebral and spinal cord defects within the same animal. The major advantage of high resolution micro-CT is the ability to provide detailed images of the anatomy that can be visualized in both 2D and 3D and enables quantitative digital analyses. Micro-CT is a highly versatile imaging method, and with appropriate contrast stains can produce high-quality images of embryo at wide range of developmental stages (Metscher, 2011), which makes microCT an attractive technique for analysis of MMC defects at early embryonic stages. Morphometric analyses at different gestation periods can provide better characteristics of the severity of bone and spinal cord damage in MMC during lesion progression. This high resolution micro-CT technique would also be relevant for quantitative measurements of MMC repair by combining anatomical data with improvement in neonatal neurologic function, which presently is difficult to study even in experimental models.
3.
Future directions
Spina bifida is one of the more common congenital malformations in humans, for which MMC is the most severe form associated with significant long-term complications (Meuli and Moehrlen, 2013; Parker et al., 2010). Despite the clinical impact of MMC, there is no effective treatment for MMC. Historically, the primary treatment for MMC, and other spinal cord disorders, was palliative, treating the symptoms and provide supportive care rather than to cure and/or modify the disease state. Currently, surgical repair of MMC before or after birth is considered to be a treatment option for some cases but still suffers from significant limitations in repairing MMC defects. However, recent research on stem cells and tissue engineering warrants the possibility of actually reversing the course of some neurologic disorders by repairing, replacing and/or regeneration of damaged cells (Thwaites et al., 2012; Wong et al., 2014). Tissue engineering holds great promise for the future treatment of birth defects present in MMC; because, the ultimate pathology in these conditions is lack of proper development or loss of tissue. Accordingly, a modest but growing body of research indicates that prenatal repair of MMC by tissue engineering approaches may be promising; however, thus far the documentation of results is very limited and it lacks important information on
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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evaluation of newly formed tissues, making interpretation difficult. For example not all studies performed histological analyses of repaired MMC and some only described histological results without the photodocumentation to support the claim (for review see (Watanabe et al., 2014)). This may stem from the fact that the assessment of new tissue formation using traditional histological techniques can be time consuming and difficult in fetuses with MMC. One of the most promising prenatal approaches for repair of MMC is the use of scaffolds in combination with stem cells and/or signaling molecules for 3D tissue formation. Efficient methodologies that provide full 3D visualization and quantitative assessment of newly formed tissues are thus necessary in order to compare the efficacy of different treatment strategies as well as establish a correlation between the anatomical repair and neurological functional outcomes in studies of MMC repair. In light of the above considerations, it appears that the use of high resolution 3D micro-CT imaging with and without contrast agents that has a potential to visualize the MMC anatomy with a micron resolution and enables quantitative digital analyses of MMC, may serve as a non-destructive alternative, or adjunct to histological methods for 3D visualization and/or quantification of MMC repair. Even though the prenatal treatment of MMC is improving with better understanding of the pathophysiology associated with MMC (Meuli and Moehrlen, 2013; Moldenhauer, 2014; Watanabe et al., 2014), it is evident that further improvements in prenatal treatment approaches and deeper understanding of MMC pathogenesis are needed. Currently, the research for prenatal treatment of MMC mainly focuses on approaches for providing soft tissue coverage of the MMC defect in gestation to prevent amniotic-fluid induced spinal cord damage and/or attempts for achieving neuroprotection and potentially spinal cord regeneration (Watanabe et al., 2014). However, it is likely that in the future the preventive strategies and the therapeutic combinations affecting also the vertebral defect will be necessary to substantially improve the MMC treatment. Owing to the important role of micro-CT in studies of bone repair and quantitative assessment of 3D tissue formation (Campbell and Sophocleous, 2014; Guldberg et al., 2008), the inclusion of 3D micro-CT imaging to complement histological evaluation of MMC repair would be essential to determine the treatment efficacy of different treatment strategies in future studies. Our earlier study provides primarily evidence for 3D visualization and quantitative evaluation of vertebral defects in animal model of MMC (Barbe et al., 2014). However, a key advantage of micro-CT is that it provides imaging with adequate resolution for the detection and measurement of bone defects and with the use of contrast agents may be used to analyze soft tissues (Campbell and Sophocleous, 2014; Cunningham and Black, 2009; Guldberg et al., 2008; Oest et al., 2008; Saito and Murase, 2013; Tahara and Larsson, 2013; Vegesna et al., 2012; Vegesna et al., 2013). Thus, further development of micro-CT assessments of MMC should allow for full 3D visualization of MMC defect e.g., the assessment of spinal cord and vertebral abnormalities in the same animal, providing an efficient methodology for quantitative morphological analysis of the severity these defects in MMC. This method of non-destructive MMC evaluation would significantly improve
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quantitative assessment of animal models of MMC and interpretation of results because destructive methods of whole-body skeleton staining and 2D analysis of spinal cord provide only discrete information and often incomplete representation of 3D environment. Besides its impact on the advance of testing prenatal treatments for MMC, the use of advanced methods such as micro-CT for a 3D visualization and quantitative assessment of bone and soft tissue damage in MMC should aid in more accurate descriptions of MMC pathology in studies on the progression of MMC during gestation in animal models. Although the microCT-system is not capable of molecular or cellular assessments as traditional methods are, but as needed, this limitation can be circumvented by the use of immunohistochemistry in conjunction with micro-CT methods (Iwasaki et al., 2012). In summary, it is evident that further improvements in prenatal and preventive MMC treatment approaches are needed to advance treatments of MMC. However, a deeper understanding of MMC pathogenesis is a prerequisite for effective preventive and therapeutic strategies. We propose that the inclusion of 3D imaging techniques such as micro-CT for visualization and quantitative measures of musculoskeletal and spinal cord defects in animal models of MMC will aid in a deeper understanding of MMC pathogenesis and provide quantitative and 3D information on repair of MMC that are essential to determine the efficacy of treatment strategies in preclinical animal models. Finally, standardized and quantitative outcome measures are needed to compare the efficacy of different treatment strategies and improve the efficiency of MMC studies.
Acknowledgments The authors thank Dr. Mary F. Barbe and colleagues from the Department of Anatomy and Cell Biology, Temple University School of Medicine for providing micro-computed tomography equipment and expertise, which was essential to the evaluation of MMC in a rat model. The authors also thank members of SHPRC for their support and sharing of ideas. Grant support from Shriners Hospitals (85230-PHI and 86400-PHI) is gratefully acknowledged.
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Thwaites, J.W., Reebye, V., Mintz, P., Levicar, N., Habib, N., 2012. Cellular replacement and regenerative medicine therapies in ischemic stroke. Regen. Med. 7, 387–395. Tibbles, L., Wiley, M.J., 1988. A comparative study of the effects of retinoic acid given during the critical period for inducing spina bifida in mice and hamsters. Teratology 37, 113–125. Tomlinson, P., Sugarman, I.D., 1995. Complications with shunts in adults with spina bifida. BMJ 311, 286–287. Vasquez, S.X., Shah, N., Hoberman, A.M., 2013. Small animal imaging and examination by micro-CT. Methods Mol. Biol. 947, 223–231. Vegesna, A.K., Sloan, J.A., Singh, B., Phillips, S.J., Braverman, A.S., Barbe, M.F., Ruggieri, M.R., Miller, L.S., S., 2013. Characterization of the distal esophagus high-pressure zone with manometry, ultrasound and micro-computed tomography. Neurogastroenterol. Motil. 25, 53–60. Vegesna, A.K., Chuang, K.Y., Besetty, R., Phillips, S.J., Braverman, A.S., Barbe, M.F., Ruggieri, M.R., Miller, L.S., 2012. Circular smooth muscle contributes to esophageal shortening during peristalsis. World J. Gastroenterol. 18, 4317–4322. Verhoef, M., Barf, H.A., Post, M.W., van Asbeck, F.W., Gooskens, R.H., Prevo, A.J., 2004. Secondary impairments in young adults with spina bifida. Dev. Med. Child Neurol. 46, 420–427. Watanabe, M., Kim, A.G., Flake, A.W., 2014. Tissue engineering strategies for fetal myelomeningocele repair in animal models. Fetal Diagn Ther. Watanabe, M., Li, H., Roybal, J., Santore, M., Radu, A., Jo, J., Kaneko, M., Tabata, Y., Flake, A., 2011. A tissue engineering approach for prenatal closure of myelomeningocele: comparison of gelatin sponge and microsphere scaffolds and bioactive protein coatings. Tissue Eng. Part A. 17, 1099–1110. Wong, F.S., Chan, B.P., Lo, A.C., 2014. Carriers in cell-based therapies for neurological disorders. Int. J. Mol. Sci. 15, 10669–10723. Zhao, J.J., Sun, D.G., Wang, J., Liu, S.R., Zhang, C.Y., Zhu, M.X., Ma, X., 2008. Retinoic acid downregulates microRNAs to induce abnormal development of spinal cord in spina bifida rat model. Childs Nerv. Syst. 24, 485–492.
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Research Report
Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida George M. Smitha,b, Barbara Krynskaa,c,n a
Shriners Hospitals Pediatric Research Center and Center for Neural Repair and Rehabilitation, Temple University School of Medicine, Philadelphia, PA 19140, USA b Department of Neuroscience, Temple University School of Medicine Philadelphia, PA 19140, USA c Department of Neurology, Temple University School of Medicine Philadelphia, PA 19140, USA
art i cle i nfo
ab st rac t
Article history:
Myelomeningocele (MMC) is a devastating spinal cord birth defect, which results in
Accepted 25 November 2014
significant life-long disabilities, impaired quality of life, and difficult medical management. The pathological progression of MMC involves failure in neural tube and vertebral arch
Keywords:
closure at early gestational ages, followed by subsequent impairment in spinal cord and
Myelomeningocele
vertebral growth during fetal development. MMC is irreversible at term. Thus, prenatal
Spina bifida
therapeutic strategies that interrupt progressive pathological processes offer an appealing
Birth defects
approach for treatment of MMC. However, a thorough understanding of pathological
Spinal cord injury
progression of MMC is mandatory for appropriate treatment to be rendered.
Vertebral defects
This article is part of a Special Issue entitled SI: Spinal cord injury.
Micro-computed tomography
1.
Introduction
In this review, we discuss the MMC spinal cord lesion with emphasis on the associated vertebral defects and how the use of sensitive and quantitative outcome measures can precisely identify and quantify changes in spinal cord and vertebral defects to better characterize progression and identify treatments for MMC. The accurate identification and quantitative evaluation of MMC abnormalities is challenging because MMC is a highly complex defect, characterized by a dynamic malformation process during gestation and varying degrees of neural tissue loss and vertebral defects. Recent refinements
& 2014 Elsevier B.V. All rights reserved.
in available imaging techniques can be used to precisely identify and quantitatively evaluate the defect or repair status of MMC. This review highlights the background and rationale for the use of 3D high resolution micro-computed tomography (micro-CT) imaging and digital analyses methods for improved quantitative assessment of MMC defects in animal models. We propose that the inclusion of 3D imaging and quantitative measures of MMC defects using high-resolution micro-CT will provide a deeper understanding of pathophysiology or repair strategy of MMC in animal models, and make the studies of MMC more effective and efficient.
n Correspondence to: Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3500 N Broad Street, Philadelphia, PA 19140, USA. Fax: þ1 215 707 8235. E-mail address: [email protected] (B. Krynska).
http://dx.doi.org/10.1016/j.brainres.2014.11.053 0006-8993/& 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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2.
MMC the problem
Myelomeningocele (MMC), the most severe form of spina bifida, is a common and devastating congenital malformation and a major source of disabilities and morbidity in children. MMC affects less than one of 1000 live births in the United States (Parker et al., 2010). About 75% of affected children survive to adulthood, but, live with significant life-long physical disabilities (Bowman et al., 2001). The exact cause of spina bifida is unknown; however, teratogenic, nutritional and genetic factors are associated with the development of spina bifida (Alles and Sulik, 1990; Kibar et al., 2007; Ornoy, 2009). Although certain risk factors are known to be associated with the development of spina bifida, and the prevalence of spina bifida has declined in the post-fortification era with the use of folic acid supplementation to reproductive age women, spina bifida still can affect the pregnancy of any women, at any age, or of any baseline health status (Moldenhauer, 2014; Osterhues et al., 2013). Despite the clinical impact of MMC, there is no cure for this devastating congenital defect. Children affected by MMC face significant life-long physical disabilities including, leg paralysis and sensory loss, bowel and bladder dysfunctions, skeletal deformations, and the Arnold-Chiari II malformation with secondary hydrocephalus (Dias and McLone, 1993; Hunt, 1990; Hunt and Poulton, 1995). These disabilities can lead to other medical conditions that require clinical care. The most common secondary conditions associated with MMC are urinary tract infections followed by complications from devices/ implants and various types of skin injuries e.g., pressure ulcers resulting from prolonged sitting in one position (Dicianno and Wilson, 2010; Verhoef et al., 2004). Approximately 30–50% of MMC patients rely on a wheelchair for mobility (Hunt and Oakeshott, 2003; McDonnell and McCann, 2000). The individuals living with the defect have a profoundly diminished quality of life and ability to work. They usually require lifelong support and institutional care. To optimize the quality of life and improve the functional outcomes of these individuals, lifelong multidisciplinary care including neurosurgery, urology, orthopedics and rehabilitation is needed (Sandler, 2010). Treatment and management of patients with spina bifida continue to have a huge economic burden on the health care system (Centers for Disease Control and Prevention (CDC) 2007; Radcliff et al., 2012), and its physical and emotional burden on children and their families is devastating.
2.1.
Spinal cord injury in MMC
MMC is a complex congenital defect of neural tube and vertebral arches closure, characterized by a dynamic pathological process during gestation that results in the protrusion of the spinal cord and meninges through a defect in the spine leading to the spinal cord injury at the level of MMC. The condition can result in varying sensory and motor deficits below the affected neurologic level of the lesion, and a variety of associated problems, including hindbrain herniation, hydrocephaly, loss of bowel and bladder control and various orthopedic complications (Dias and McLone, 1993; Hunt 1990; Hunt and Poulton, 1995; Hunt and Oakeshott, 2003; Tomlinson and Sugarman, 1995). A better understanding of spinal cord injury in MMC has been revised
during the last decade, resulting in the “two-hit hypothesis”. The first hit, denotes the initial defect of neurulation and the secondary hit represents the subsequent in utero acquired damage to the openly exposed spinal cord (Meuli and Moehrlen, 2013). Although, failure during neurulation is the initial cause for the spinal cord malformation, compelling clinical as well as experimental evidence suggest that the exposed spinal cord results in secondary in utero damage, predominantly causing the neurological deficit associated with MMC (Heffez et al., 1990; Meuli et al., 1995b; Sival et al., 1997; Stiefel et al., 2007). Human studies show that the openly exposed spinal cord is histologically intact in early gestation, showing little damage; however, progressive damage sometimes leading to complete loss, was observed in late stages of gestation (Hutchins et al., 1996; Meuli et al., 1997). In support of the in utero acquired secondary damage, experimental evidence demonstrated that surgically induced in utero exposure of the spinal cord (human-like spina bifida lesions) in experimental animals (rat, pig, sheep) led to spinal cord damage of the exposed segments and functional loss observed at birth (Heffez et al., 1993; Meuli et al., 1995a; Meuli et al., 1995b). The definitive secondary mechanisms underlying the pathogenesis of spinal cord injury remain unclear. Although, it has been proposed that the in utero acquired damage of the spinal cord includes, mechanical, degenerative, and possibly inflammatory damage to the openly exposed spinal cord tissue caused by exposure to amniotic fluid, traumatic injury within the amniotic space, hydrodynamic pressure, or a combination of these (Heffez et al., 1993; Meuli et al., 1995a; Meuli et al., 1995b; Meuli and Moehrlen, 2013). Thus, prenatal therapeutic strategies that interrupt the pathological processes offer an appealing approach for treatment of MMC.
2.2.
Prenatal and postnatal repair for MMC
Despite the clinical impact of MMC, there is no cure for this devastating defect. Prevention strategies can reduce the onset of MMC; however, uncertainty remains regarding its etiology and the underlying mechanisms, which is a prerequisite for effective preventive strategies. Prophylaxis treatment with folic acid has proven efficacious in reducing total numbers, however, it helps only a fraction of pregnant women, and thus a significant portion of neural tube defects still persists (Oakley, 2010; Osterhues et al., 2013; Parker et al., 2010). Once spina bifida occurs folic acid is not helpful. Therefore, women carrying a fetus diagnosed with MMC have few options, including standard surgical closure shortly after birth, closure of the defect during the fetal period in selected cases, or termination of pregnancy. Postnatal surgical closure of MMC defect is associated with poor neurological outcomes because the postnatal therapy cannot prevent or reverse the neurological injury, since the affected spinal cord is destroyed and neurologic function lost before birth (Adzick et al., 2011; Simpson and Greene, 2011). Because experimental and clinical evidence shows progressive damage to the defective area during fetal development in utero, prenatal surgical closure was thought to be a potential treatment (Heffez et al., 1990; Hutchins et al., 1996; Meuli et al., 1995b; Meuli et al., 1997; Sival et al., 1997; Stiefel et al., 2007). Indeed, animal experiments of in utero repair after surgically created defects showed improved neural function (Heffez et al., 1993; Meuli et al., 1996).
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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Given that prenatal diagnosis of neural tube defects and surgical techniques have improved over the past several years, the concept of in utero repair of MMC in humans became feasible. In utero surgical closure of MMC improves the functional neurological outcomes including the ability to walk without orthotics or devices in some patients, a reduction in hindbrain herniation and a decreased need for ventriculoperitoneal shunting in patients who underwent prenatal compared with those who had postnatal closure (Adzick et al., 2011; Simpson and Greene, 2011). Currently, more than ten centers in the United States are performing in utero MMC repair, which now is a clinical option for the management of prenatally diagnosed MMC in eligible patients (Moldenhauer, 2014). However, despite the supporting evidence that the prenatal surgical repair is more successful than postnatal MMC repair, the surgical procedure occur well after the initial exposure of the spinal cord to the amniotic environment, and does not completely repair the spinal cord injury, so significant morbidity remains in most patients. In addition, there are significant maternal and fetal risks associated with prenatal surgery and the impact on the long-term outcomes of children who underwent fetal surgery for MMC is currently unknown and requires further study (Moldenhauer, 2014). Thus, further research of this defect is needed to advance the understanding of MMC development and identify new treatment approaches, through which the concept of preventing or reversing neurological impairments caused by MMC may be achieved.
2.3.
Vertebral (mesenchymal) defects in MMC
Spinal cord injury, which occurs with MMC is thought to be a progressive intrauterine phenomenon, rather than completely a condition induced by defective neurulation. The etiology and pathogenesis of MMC is unclear and most likely mulifactorial. The accepted view has been that the neuroepithelium is initially involved in the failure of neural tube closure and that the associated vertebral (mesenchymal) anomalies are secondary (Copp and Greene, 2013; Finnell et al., 2000). While spina bifida is often synonymous with neural tube defects, other etiologies have also been proposed for spina bifida development. Analysis of mouse models of spina bifida suggests that the mesoderm surrounding the neural tube may be initially involved in development of this defect (Helwig et al., 1995; Payne et al., 1997; Pickett et al., 2008; Takahashi et al., 1992). In experimental systems MMClike defects can be induced by retinoic acid overexposure (Alles and Sulik, 1990; Danzer et al., 2005; Tibbles and Wiley, 1988). Although the exact mechanisms through which alltrans retinoic acid induces MMC remains largely unknown, it has been proposed that excessive cell death of embryonic mesenchymal cells may play a role in the induction of these malformations (Alles and Sulik, 1990). Early studies on human fetuses with MMC suggest that the musculoskeletal system may be initially affected resulting in open exposure of the spinal cord, followed by progressive in utero damage to the spinal cord (Hutchins et al., 1992; Jordan et al., 1991). These findings support the hypothesis that some categories of human MMC may arise from a failure of the vertebrae to close at the midline, suggesting that the primary abnormality may be in the musculoskeletal system. Accordingly, animal
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models of MMC created by fetal surgery provide evidence that open exposure of the normal spinal cord to the intrauterine environment results in MMC-type lesion that have clinical and morphological similarities to human MMC (Heffez et al., 1990; Hutchins et al., 1996; Meuli et al., 1995b). Surgical lesions that remove the vertebral arches without directly damaging the spinal cord resulted in herniation of the spinal cord and its membranes through the vertebral defect, forming a cystic human-like MMC (Meuli et al., 1995b). In addition, earlier studies in primates also show that MMC created by fetal laminectomy resulted in the spinal cord injury and MMClike neurologic deficits at birth (Michejda, 1984). Immediate in utero repair of the bone deficit after fetal laminectomy resulted in these animals having near normal neurological scores at birth (Michejda, 1984). Although, several etiologies have been proposed for spina bifida, all with unknown casual mechanisms, it appears that the sentinel pathology in MMC is in the defect in the mesodermallyderived musculoskeletal tissues. The neurologic injury occurs secondary to these defects. Convincing experimental and clinical evidence indicates no signs of trauma or degeneration of spinal tissue at early gestational time points, and spinal cord degeneration appears to progress in severity with gestational age (Keller-Peck and Mullen, 1996; Meuli et al., 1997; Selcuki et al., 2001; Stiefel et al., 2007). With naturally occurring MMC, part of the spinal cord pushes through congenitally unclosed openings in overlying vertebrae and protrudes from the fetus' back into amniotic space, followed by progressive damage to the spinal cord leading in some cases to complete loss observed in late stages of gestation (Hutchins et al., 1996; Meuli et al., 1997). The extent of neurological deficits and disabilities associated with MMC is dependent on the severity of the defect. Although, the intrauterine conditions are thought to negatively impact spinal cord development leading to functional loss observed at birth, there is little experimental data, e.g., from animal models for the quantitative assessment of MMC pathology. Given the nature of the MMC defect, one potential quantitative means of assessing defect severity during gestation is by measuring impairment of fetal vertebral development with respect to the degree of spinal cord damage, at different gestation periods. In combination with neurological assessment of animals, the information obtained from these studies would establish a correlation between abnormal fetal spine development, the spinal cord injury and loss of neurologic function before birth. A comprehensive quantitative analysis of MMC pathology would improve an assessment of MMC, while measurements at different gestation periods should advance the understanding of the developmental trajectory of MMC.
2.4.
Quantitative assessment of MMC in an animal model
Although several animal models of MMC have been developed to study the pathogenesis and treatment approaches, limited work has been done to improve the quantitative outcome measures of MMC. At present, anatomical MMC assessment in animal models is usually performed by conventional 2D histopathological analysis of spinal cord defect and whole-body skeleton staining (Cai et al., 2007; Danzer et al., 2005). While histological methods offer unique capabilities, these traditional techniques may be inadequate to fully visualize and
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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quantitatively evaluate MMC defects. The methods of wholebody skeleton staining are invasive, destructive techniques that severely limit precise 3D visualization and quantitative assessment of skeletal defects in MMC fetuses and are time consuming. The invasiveness and 2D analyses of histological sections are also the main disadvantages limiting the opportunity for full 3D visualization and quantitative evaluation of fetal spinal cord abnormalities in MMC. In addition, the complete assessment of MMC defects can be difficult to perform in the same animal using traditional techniques. For example, due to the destructive nature of histological techniques, analysis of the vertebral level and skin level of the MMC lesion in mice after whole skeletal preparations was performed using the correlation of the cranial end of the skeletal defect with the suturemarked cranial end of the skin defect (Stiefel and Meuli, 2008). Advanced techniques, e.g. MRI can provide 3D images of MMC in animal model (Danzer et al., 2005) however, data acquisition is usually time-consuming and it is difficult to obtain highresolution imaging in small animals adequate for quantitative analysis of MMC, in particular the skeletal defect. Micro-computed tomography (micro-CT) is a non-invasive whole volume imaging technique that provides high contrast between soft tissue and bone and offers quantitative data on bone morphology and architecture and has been helpful in characterizing developmental defects of bone (Bouxsein et al., 2010; Ford-Hutchinson et al., 2003; Oest et al., 2008; Vasquez et al., 2013). The great detail with which the anatomy can be visualized and the abnormalities quantified (Cunningham and Black, 2009; Oest et al., 2008), makes high resolution micro-CT an attractive imaging technique for assessing the characteristics of MMC skeletal defects in animal models. As an initial step toward the development of improved methods for assessment of MMC, we have recently described the assessment of MMC skeletal defects in an established retinoic acid induced rat model of lumbosacral MMC using high-resolution micro-CT imaging combined with digital quantification methods (Barbe et al., 2014). The retinoic acid induced rat model of MMC is developmentally and phenotypically analogous to human MMC, and is relevant for investigating the pathogenesis and in utero treatments of MMC (Danzer et al., 2005; Danzer et al., 2011; Watanabe et al., 2011; Zhao et al., 2008). Using 3D micro-CT images of rat vertebral defects and corresponding normal vertebra we observed, in detail, skeletal defects in lumbosacral vertebra of MMC rats, including morphological defects of individual dorsal vertebral arches. The defect in lambosacral vertebra appeared in the form of delayed or absent ossification. Butterfly-shaped, dumbbellshaped, non-fused, and absent to nearly absent vertebra centra were identified using high resolution 3D micro-CT images of lumbosacral vertebrae (Barbe et al., 2014). We show highresolution micro-CT to be an especially powerful method of analysis that provides not only anatomical details of MMC defect, but also adequate 2D and 3D images for digital quantification not previously achieved in models of MMC. We were able to nondestructively quantify the distance between the ends of the vertebral arches in the retinoic acid induced rat model of MMC and showed a significant increase in the distance between dorsal vertebral arches from L5 to S4 when compared to the control group. In addition, micro-CT method is a powerful tool for assessing overall skeletal phenotype and it allowed us to visualize supernumerary ribs in fetuses with MMC. As often
reported in the literature, micro-CT is the imaging technique of choice for the measurement and visualization of bone structure. It can also be used for quantitative analysis of various fetal skeletal development parameters including bone mineral density as well as identification of skeletal malformations with high degree of accuracy (Campbell and Sophocleous, 2014; Cunningham and Black, 2009; Oest et al., 2008). Micro CT has also become a popular imaging technique to study soft tissues (Campbell and Sophocleous, 2014). Recent, studies indicate that ex vivo micro-CT imaging, with the aid of staining agents that make soft tissues radiopaque, has the potential for non-invasive imaging and quantitative analysis of soft tissues in 3D including spinal cord (Saito and Murase, 2013; Tahara and Larsson, 2013; Vegesna et al., 2012; Vegesna et al., 2013). Although, the optimization of staining methods is needed to distinguish soft tissue from bone tissue and delineate spinal cord, these studies indicate the potential of micro-CT for evaluation of both vertebral and spinal cord defects within the same animal. The major advantage of high resolution micro-CT is the ability to provide detailed images of the anatomy that can be visualized in both 2D and 3D and enables quantitative digital analyses. Micro-CT is a highly versatile imaging method, and with appropriate contrast stains can produce high-quality images of embryo at wide range of developmental stages (Metscher, 2011), which makes microCT an attractive technique for analysis of MMC defects at early embryonic stages. Morphometric analyses at different gestation periods can provide better characteristics of the severity of bone and spinal cord damage in MMC during lesion progression. This high resolution micro-CT technique would also be relevant for quantitative measurements of MMC repair by combining anatomical data with improvement in neonatal neurologic function, which presently is difficult to study even in experimental models.
3.
Future directions
Spina bifida is one of the more common congenital malformations in humans, for which MMC is the most severe form associated with significant long-term complications (Meuli and Moehrlen, 2013; Parker et al., 2010). Despite the clinical impact of MMC, there is no effective treatment for MMC. Historically, the primary treatment for MMC, and other spinal cord disorders, was palliative, treating the symptoms and provide supportive care rather than to cure and/or modify the disease state. Currently, surgical repair of MMC before or after birth is considered to be a treatment option for some cases but still suffers from significant limitations in repairing MMC defects. However, recent research on stem cells and tissue engineering warrants the possibility of actually reversing the course of some neurologic disorders by repairing, replacing and/or regeneration of damaged cells (Thwaites et al., 2012; Wong et al., 2014). Tissue engineering holds great promise for the future treatment of birth defects present in MMC; because, the ultimate pathology in these conditions is lack of proper development or loss of tissue. Accordingly, a modest but growing body of research indicates that prenatal repair of MMC by tissue engineering approaches may be promising; however, thus far the documentation of results is very limited and it lacks important information on
Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
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evaluation of newly formed tissues, making interpretation difficult. For example not all studies performed histological analyses of repaired MMC and some only described histological results without the photodocumentation to support the claim (for review see (Watanabe et al., 2014)). This may stem from the fact that the assessment of new tissue formation using traditional histological techniques can be time consuming and difficult in fetuses with MMC. One of the most promising prenatal approaches for repair of MMC is the use of scaffolds in combination with stem cells and/or signaling molecules for 3D tissue formation. Efficient methodologies that provide full 3D visualization and quantitative assessment of newly formed tissues are thus necessary in order to compare the efficacy of different treatment strategies as well as establish a correlation between the anatomical repair and neurological functional outcomes in studies of MMC repair. In light of the above considerations, it appears that the use of high resolution 3D micro-CT imaging with and without contrast agents that has a potential to visualize the MMC anatomy with a micron resolution and enables quantitative digital analyses of MMC, may serve as a non-destructive alternative, or adjunct to histological methods for 3D visualization and/or quantification of MMC repair. Even though the prenatal treatment of MMC is improving with better understanding of the pathophysiology associated with MMC (Meuli and Moehrlen, 2013; Moldenhauer, 2014; Watanabe et al., 2014), it is evident that further improvements in prenatal treatment approaches and deeper understanding of MMC pathogenesis are needed. Currently, the research for prenatal treatment of MMC mainly focuses on approaches for providing soft tissue coverage of the MMC defect in gestation to prevent amniotic-fluid induced spinal cord damage and/or attempts for achieving neuroprotection and potentially spinal cord regeneration (Watanabe et al., 2014). However, it is likely that in the future the preventive strategies and the therapeutic combinations affecting also the vertebral defect will be necessary to substantially improve the MMC treatment. Owing to the important role of micro-CT in studies of bone repair and quantitative assessment of 3D tissue formation (Campbell and Sophocleous, 2014; Guldberg et al., 2008), the inclusion of 3D micro-CT imaging to complement histological evaluation of MMC repair would be essential to determine the treatment efficacy of different treatment strategies in future studies. Our earlier study provides primarily evidence for 3D visualization and quantitative evaluation of vertebral defects in animal model of MMC (Barbe et al., 2014). However, a key advantage of micro-CT is that it provides imaging with adequate resolution for the detection and measurement of bone defects and with the use of contrast agents may be used to analyze soft tissues (Campbell and Sophocleous, 2014; Cunningham and Black, 2009; Guldberg et al., 2008; Oest et al., 2008; Saito and Murase, 2013; Tahara and Larsson, 2013; Vegesna et al., 2012; Vegesna et al., 2013). Thus, further development of micro-CT assessments of MMC should allow for full 3D visualization of MMC defect e.g., the assessment of spinal cord and vertebral abnormalities in the same animal, providing an efficient methodology for quantitative morphological analysis of the severity these defects in MMC. This method of non-destructive MMC evaluation would significantly improve
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quantitative assessment of animal models of MMC and interpretation of results because destructive methods of whole-body skeleton staining and 2D analysis of spinal cord provide only discrete information and often incomplete representation of 3D environment. Besides its impact on the advance of testing prenatal treatments for MMC, the use of advanced methods such as micro-CT for a 3D visualization and quantitative assessment of bone and soft tissue damage in MMC should aid in more accurate descriptions of MMC pathology in studies on the progression of MMC during gestation in animal models. Although the microCT-system is not capable of molecular or cellular assessments as traditional methods are, but as needed, this limitation can be circumvented by the use of immunohistochemistry in conjunction with micro-CT methods (Iwasaki et al., 2012). In summary, it is evident that further improvements in prenatal and preventive MMC treatment approaches are needed to advance treatments of MMC. However, a deeper understanding of MMC pathogenesis is a prerequisite for effective preventive and therapeutic strategies. We propose that the inclusion of 3D imaging techniques such as micro-CT for visualization and quantitative measures of musculoskeletal and spinal cord defects in animal models of MMC will aid in a deeper understanding of MMC pathogenesis and provide quantitative and 3D information on repair of MMC that are essential to determine the efficacy of treatment strategies in preclinical animal models. Finally, standardized and quantitative outcome measures are needed to compare the efficacy of different treatment strategies and improve the efficiency of MMC studies.
Acknowledgments The authors thank Dr. Mary F. Barbe and colleagues from the Department of Anatomy and Cell Biology, Temple University School of Medicine for providing micro-computed tomography equipment and expertise, which was essential to the evaluation of MMC in a rat model. The authors also thank members of SHPRC for their support and sharing of ideas. Grant support from Shriners Hospitals (85230-PHI and 86400-PHI) is gratefully acknowledged.
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Please cite this article as: Smith, G.M., Krynska, B., Myelomeningocele: How we can improve the assessment of the most severe form of spina bifida. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.11.053
