Three-dimensional optical coherence tomography of whole-muscle autografts as a precursor to morphological assessment of muscular dystrophy in mice

Three-dimensional optical coherence tomography (3D-OCT) is used to evaluate the structure and pathology of regenerating mouse skeletal muscle autografts for the first time. The death of myofibers with associated inflammation and subsequent new muscle formation in this graft model represents key features of necrosis and inflammation in the human disease Duchenne muscular dystrophy. We perform 3D-OCT imaging of excised autografts and compare OCT images with coregistered histology. The OCT images readily distinguish the necrotic and inflammatory tissue of the graft from the intact healthy muscle fibers in the underlying host tissue. These preliminary findings suggest that, with further development, 3D-OCT could be used as a tool for the evaluation of small-animal muscle morphology and pathology, in particular, for analysis of mouse models of muscular dystrophy.     

In situ imaging of lung alveoli with an optical coherence tomography needle probe

In situ imaging of alveoli and the smaller airways with optical coherence tomography (OCT) has significant potential in the assessment of lung disease. We present a minimally invasive imaging technique utilizing an OCT needle probe. The side-facing needle probe comprises miniaturized focusing optics consisting of no-core and GRIN fiber encased within a 23-gauge needle. 3D-OCT volumetric data sets were acquired by rotating and retracting the probe during imaging. The probe was used to image an intact, fresh (not fixed) sheep lung filled with normal saline, and the results validated against a histological gold standard. We present the first published images of alveoli acquired with an OCT needle probe and demonstrate the potential of this technique to visualize other anatomical features such as bifurcations of the bronchioles.     

Combined multiplanar optical coherence tomography and confocal scanning ophthalmoscopy

We demonstrate the clinical application of a multiplanar imaging system that simultaneously acquires en face (C-scan) optical coherence tomography (OCT) and the corresponding confocal ophthalmoscopic images, along with cross-sectional (B-scan) OCT at specifiable locations on the confocal image. The advantages of the simultaneous OCT and confocal acquisition as well as the challenges of interpreting the C-scan OCT images are discussed. Variations in tissue inclination with respect to the coherence wave surface alter the sampling of structures within the depth of the retina, producing novel slice orientations that are often challenging to interpret. We have evaluated for the first time the utility of C-scan OCT for a variety of pathologies, including melanocytoma, diabetic retinopathy, choroidal neovascular membrane, and macular pucker. Several remarkable new aspects of clinical anatomy were revealed using this new technique. The versatility of selective capture of C-scan OCT images and B-scan OCT images at precise points on the confocal image affords the clinician a more complete and interactive tool for 3-D imaging of retinal pathology.     

Effect of tissue preservation on imaging using ultrahigh resolution optical coherence tomography

Ultrahigh resolution optical coherence tomography (OCT) is an emerging imaging modality that enables noninvasive imaging of tissue with 1- to 3-microm resolutions. Initial OCT studies have typically been performed using harvested tissue specimens (ex vivo). No reports have investigated postexcision tissue degradation on OCT image quality. We investigate the effects of formalin fixation and commonly used cell culture media on tissue optical scattering characteristics in OCT images at different times postexcision compared to in vivo conditions. OCT imaging at 800-nm wavelength with 1.5-mum axial resolution is used to image the hamster cheek pouch in vivo, followed by excision and imaging during preservation in phosphate-buffered saline (PBS), Dulbecco's Modified Eagle's Media (DMEM), and 10% neutral-buffered formalin. Imaging is performed in vivo and at sequential time points postexcision from 15 min to 10 to 18 h. Formalin fixation results in increases in scattering intensity from the muscle layers, as well as shrinkage of the epithelium, muscle, and connective tissue of approximately 50%. PBS preservation shows loss of optical contrast within two hours, occurring predominantly in deep muscle and connective tissue. DMEM maintains tissue structure and optical scattering characteristics close to in vivo conditions up to 4 to 6 h after excision and best preserved tissue optical properties when compared to in vivo imaging.     

Computer recognition of cancer in the urinary bladder using optical coherence tomography and texture analysis

The vast majority of bladder cancers originate within 600 microm of the tissue surface, making optical coherence tomography (OCT) a potentially powerful tool for recognizing cancers that are not easily visible with current techniques. OCT is a new technology, however, and surgeons are not familiar with the resulting images. Technology able to analyze and provide diagnoses based on OCT images would improve the clinical utility of OCT systems. We present an automated algorithm that uses texture analysis to detect bladder cancer from OCT images. Our algorithm was applied to 182 OCT images of bladder tissue, taken from 68 distinct areas and 21 patients, to classify the images as noncancerous, dysplasia, carcinoma in situ (CIS), or papillary lesions, and to determine tumor invasion. The results, when compared with the corresponding pathology, indicate that the algorithm is effective at differentiating cancerous from noncancerous tissue with a sensitivity of 92% and a specificity of 62%. With further research to improve discrimination between cancer types and recognition of false positives, it may be possible to use OCT to guide endoscopic biopsies toward tissue likely to contain cancer and to avoid unnecessary biopsies of normal tissue.     

Temperature-dependent optical properties of individual vascular wall components measured by optical coherence tomography

Optical properties of tissues and tissue components are important parameters in biomedical optics. We report measurements of tissue refractive index n and the attenuation coefficient mu(t) using optical coherence tomography (OCT) of individual vascular wall layers and plaque components. Moreover, since the temperature dependence of optical properties is widely known, we compare measurements at room and body temperatures. A decrease of n and mu(t) is observed in all samples, with the most profound effect on samples with high lipid content. The sample temperature is of influence on the quantitative measurements within OCT images. For extrapolation of ex-vivo experimental results, especially for structures with high lipid content, this effect should be taken into account.     

Extracting structural features of rat sciatic nerve using polarization-sensitive spectral domain optical coherence tomography

We present spectral domain polarization-sensitive optical coherence tomography (SD PS-OCT) imaging of peripheral nerves. Structural and polarization-sensitive OCT imaging of uninjured rat sciatic nerves was evaluated both qualitatively and quantitatively. OCT and its functional extension, PS-OCT, were used to image sciatic nerve structure with clear delineation of the nerve boundaries to muscle and adipose tissues. A long-known optical effect, bands of Fontana, was also observed. Postprocessing analysis of these images provided significant quantitative information, such as epineurium thickness, estimates of extinction coefficient and birefringence of nerve and muscle tissue, frequency of bands of Fontana at different stretch levels of nerve, and change in average birefringence of nerve under stretched condition. We demonstrate that PS-OCT combined with regular-intensity OCT (compared with OCT alone) allows for a clearer determination of the inner and outer boundaries of the epineurium and distinction of nerve and muscle based on their birefringence pattern. PS-OCT measurements on normal nerves show that the technique is promising for studies on peripheral nerve injury.     

Transgenic overexpression of ADAM12 suppresses muscle regeneration and aggravates dystrophy in aged mdx mice

Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy.     

Overexpression of the cytotoxic T cell (CT) carbohydrate inhibits muscular dystrophy in the dyW mouse model of congenital muscular dystrophy 1A

A number of recent studies have demonstrated therapeutic effects of transgenes on the development of muscle pathology in the mdx mouse model for Duchenne muscular dystrophy, but none have been shown also to be effective in mouse models for laminin alpha2-deficient congenital muscular dystrophy (MDC1A). Here, we show that overexpression of the cytotoxic T cell (CT) GalNAc transferase (Galgt2) is effective in inhibiting the development of muscle pathology in the dy(W) mouse model of MDC1A, much as we had previously shown in mdx animals. Embryonic overexpression of Galgt2 in skeletal muscles using transgenic mice or postnatal overexpression using adeno-associated virus both reduced the extent of muscle pathology in dy(W)/dy(W) skeletal muscle. As with mdx mice, embryonic overexpression of the Galgt2 transgene in dy(W)/dy(W) myofibers inhibited muscle growth, whereas postnatal overexpression did not. Both embryonic and postnatal overexpression of Galgt2 in dy(W)/dy(W) muscle increased the expression of agrin, a protein that, in recombinant form, has been shown to ameliorate disease, whereas laminin alpha1, another disease modifier, was not expressed. Galgt2 over-expression also stimulated the glycosylation of a gly-colipid with the CT carbohydrate, and glycolipids accounted for most of the CT-reactive material in postnatal overexpression experiments. These experiments demonstrate that Galgt2 overexpression is effective in altering disease progression in skeletal muscles of dy(W) mice and should be considered as a therapeutic target in MDC1A.     

Combined deficiency of alpha and epsilon sarcoglycan disrupts the cardiac dystrophin complex

Cardiomyopathy is a puzzling complication in addition to skeletal muscle pathology for patients with mutations in β-, γ- or δ-sarcoglycan (SG) genes. Patients with mutations in α-SG rarely have associated cardiomyopathy, or their cardiac pathology is very mild. We hypothesize that a fifth SG, ε-SG, may compensate for α-SG deficiency in the heart. To investigate the function of ε-SG in striated muscle, we generated an Sgce-null mouse and a Sgca-;Sgce-null mouse, which lacks both α- and ε-SGs. While Sgce-null mice showed a wild-type phenotype, with no signs of muscular dystrophy or heart disease, the Sgca-;Sgce-null mouse developed a progressive muscular dystrophy and a more anticipated and severe cardiomyopathy. It shows a complete loss of residual SGs and a strong reduction in both dystrophin and dystroglycan. Our data indicate that ε-SG is important in preventing cardiomyopathy in α-SG deficiency.     

Detecting glucose-induced changes in in vitro and in vivo experiments with optical coherence tomography

Optical clearing is a well-known phenomenon. It is based on the matching of refractive indices of a bulk material and scattering particles. The same principle is also used in scattering-based optical measurements of different constituents, such as glucose. By registering changes in scattering, it is possible to evaluate changes in the concentration of a solvent. This work describes the use of optical coherence tomography (OCT) to monitor glucose-induced changes in the optical properties of samples. Intralipid, mouse skin tissue, and mice (C57BL) are used as samples. Differences between in vitro and in vivo measurement conditions, the effect of glucose on the samples' optical properties, as well as possible problems in OCT experiments are discussed. A comparison of OCT signals from mouse skin samples and mice in vivo shows that the intensity of backscattered radiation is stronger in a living animal than in cultured tissue. Moreover, the effect of glucose on the scattering properties is larger in an in vivo case than in an in vitro case. In comparison with tissue, the effect of glucose is the smallest in Intralipid. The results increase the value of using cultured tissue in developing optical sensing techniques.     

Use of optical coherence tomography to monitor biological tissue freezing during cryosurgery

The use of optical coherence tomography (OCT) for imaging skin during cryosurgery is evaluated. OCT provides high spatial resolution (5-10 microm) images of optical backscattering due to local variations in refractive index, such as the boundary between liquid and frozen water in tissue. Time resolved OCT images were acquired during freezing of water, Intralipid trade mark, and in vivo hamster skin. Subsurface morphological changes were evident only during freezing of Intralipid and skin. A simple thermal model was applied which predicted freezing times on the same order of magnitude as those observed in OCT images. OCT can be used as a feedback tool during cryosurgical procedures to monitor progression of the freezing front.     

免疫荧光检测抗肌萎缩蛋白诊断肌营养不良症的临床应用

目的 采用免疫荧光技术对Duchenne型肌营养不良症（Duchenne muscular dystrophy,DMD),Becker型肌营养不良症（Becker muscular dystrophy,BMD)，面肩肱型肌营养不良症（facioscapulohumeral muscular dystrophy,FSHD)以及神经性肌萎缩患者骨骼肌细胞膜的dystrophin蛋白进行检测，为临床诊断、分类肌营养不良症提供简便的实验方法。方法 对47例患者选择3种dystrophin 的鼠抗单克隆抗体、羊抗和兔抗多克隆抗体，分别进行免疫荧光技术检测。结果 16例DMD患者均为阴性染色；11例BMD患者为弱阳性染色；10例FSHD和10例神经性肌萎缩患者均为阳性染色。结论 检测肌营养不良症患者骨骼肌膜dystrophin蛋白，有助于肌营养不良症的临床诊断和分型。     

Contribution of oxidative stress to pathology in diaphragm and limb muscles with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease that makes walking and breathing difficult. DMD is caused by an X-linked (Xp21) mutation in the dystrophin gene. Dystrophin is a scaffolding protein located in the sarcolemmal cytoskeleton, important in maintaining structural integrity and regulating muscle cell (muscle fiber) growth and repair. Dystrophin deficiency in mouse models (e.g., mdx mouse) destabilizes the interface between muscle fibers and the extracellular matrix, resulting in profound damage, inflammation, and weakness in diaphragm and limb muscles. While the link between dystrophin deficiency with inflammation and pathology is multi-factorial, elevated oxidative stress has been proposed as a central mediator. Unfortunately, the use of non-specific antioxidant scavengers in mouse and human studies has led to inconsistent results, obscuring our understanding of the importance of redox signaling in pathology of muscular dystrophy. However, recent studies with more mechanistic approaches in mdx mice suggest that NAD(P)H oxidase and nuclear factor-kappaB are important in amplifying dystrophin-deficient muscle pathology. Therefore, more targeted antioxidant therapeutics may ameliorate damage and weakness in human population, thus promoting better muscle function and quality of life. This review will focus upon the pathobiology of dystrophin deficiency in diaphragm and limb muscle primarily in mouse models, with a rationale for development of targeted therapeutic antioxidants in DMD patients.     

Investigation of optical coherence tomography as an imaging modality in tissue engineering

Monitoring cell profiles in 3D porous scaffolds presents a major challenge in tissue engineering. In this study, we investigate optical coherence tomography (OCT) as an imaging modality to monitor non-invasively both structures and cells in engineered tissue constructs. We employ time-domain OCT to visualize macro-structural morphology, and whole-field optical coherence microscopy to delineate the morphology of cells and constructs in a developing in vitro engineered bone tissue. The results show great potential for the use of OCT in non-invasive monitoring of cellular activities in 3D developing engineered tissues.     

Optical coherence photoacoustic microscopy: accomplishing optical coherence tomography and photoacoustic microscopy with a single light source

We developed optical coherence photoacoustic microscopy (OC-PAM) to demonstrate that the functions of optical coherence tomography (OCT) and photoacoustic microscopy (PAM) can be achieved simultaneously by using a single illuminating light source. We used a pulsed broadband laser centered at 580 nm and detected the absorbed photons through photoacoustic detection and the back-scattered photons with an interferometer. In OC-PAM, each laser pulse generates both one OCT A-line and one PAM A-line simultaneously; as a result, the two imaging modalities are intrinsically co-registered in the lateral directions. In vivo images of the mouse ear were acquired to demonstrate the capabilities of OC-PAM.     

Transgenic overexpression of caveolin-3 in the heart induces a cardiomyopathic phenotype

Caveolins are structural protein components of caveolar membrane domains. Caveolin-3, a muscle-specific member of the caveolin family, is expressed in skeletal muscle tissue and in the heart. The multiple roles that caveolin-3 plays in cellular physiology are becoming more apparent. We have shown that lack of caveolin-3 expression in skeletal muscle resembles limb-girdle muscular dystrophy-1C. In contrast, we have demonstrated that overexpression of caveolin-3 in skeletal muscle tissue promotes defects similar to those seen in Duchenne muscular dystrophy (DMD). Thus, a tight regulation of caveolin-3 expression is fundamental for normal muscle functions. Since caveolin-3 is also endogenously expressed in cardiac myocytes, and cardiomyopathies are observed in DMD patients, we looked at the effects of overexpression of caveolin-3 on cardiac structure and function by characterizing caveolin-3 transgenic mice. Our results indicate that overexpression of caveolin-3 causes severe cardiac tissue degeneration, fibrosis and a reduction in cardiac functions. We also show that dystrophin and its associated glycoproteins are down-regulated in caveolin-3 transgenic heart. In addition, we demonstrate that the activity of nitric oxide synthase (NOS) is down-regulated by high levels of caveolin-3 in the heart. Taken together, these results indicate that overexpression of caveolin-3 is sufficient to induce severe cardiomyopathy. In addition, these findings suggest that caveolin-3 transgenic mice may represent a valid mouse model for studying the molecular mechanisms underlying cardiomyopathies associated with Duchenne muscular dystrophy.     

Speckle reduction in optical coherence tomography by "path length encoded" angular compounding

Speckle, the dominant factor reducing image quality in optical coherence tomography (OCT), limits the ability to identify cellular structures that are essential for diagnosis of a variety of diseases. We describe a new high-speed method for implementing angular compounding by path length encoding (ACPE) for reducing speckle in OCT images. By averaging images obtained at different incident angles, with each image encoded by path length, ACPE maintains high-speed image acquisition and requires minimal modifications to OCT probe optics. ACPE images obtained from tissue phantoms and human skin in vivo demonstrate a qualitative improvement over traditional OCT and an increased SNR that correlates well with theory.     

Novel algorithm of processing optical coherence tomography images for differentiation of biological tissue pathologies

A numerical algorithm based on a small-angle approximation of the radiative transfer equation (RTE) is developed to reconstruct scattering characteristics of biological tissues from optical coherence tomography (OCT) images. According to the algorithm, biological tissue is considered to be a layered random medium with a set of scattering parameters in each layer: total scattering coefficient, variance of a small-angle scattering phase function, and probability of backscattering, which fully describe the OCT signal behavior versus probing depth. The reconstruction of the scattering parameters is performed by their variation to fit the experimental OCT signal by the theoretical one using a time-saving genetic algorithm. The proposed reconstruction procedure is tested on model media with known scattering parameters. The possibility to estimate scattering parameters from OCT images is studied for various regimes of OCT signal decay. The developed algorithm is applied to reconstruct optical characteristics of epithelium and stroma for normal cervical tissue and its pathologies, and the potential to distinguish between the types of pathological changes in epithelial tissue by its OCT images is demonstrated.