Textural Analysis of a Fibronectin Network During Early Embryogenesis

Fibronectin is a major extracellular matrix molecular component that plays a critical role in embryonic cell motility and morphogenesis. During early development, the fibronectin molecular network is nearly ubiquitous in distribution across the entire embryonic volume. As a result, the embryonic fibronectin distribution is functionally relevant to both cell motility and organogenesis. Despite its biological importance, the structural attributes of embryonic fibronectin distribution are poorly understood. The textural features of an extracellular matrix network like that of fibronectin is not known. By marking the embryonic tissue, obtained during specific stages of development, using fibronectin indirect immunofluorescence, fluorescent microscopy images capturing the embryonic fibronectin distribution were obtained. Using the properties of the gray scale co-occurrence matrix, textural attributes of a fibronectin network was derived for quail embryos during their early stages of morphogenesis. As a result, textural properties like inertia, correlation, uniformity, entropy and homogeneity were assessed for the medial (including the embryonic anteroposterior axis) and lateral (excluding the embryonic anteroposterior axis) fibronectin . The results not only demonstrated a noticeable heterogeneity of fibronectin textural properties across the embryonic regions examined, but also across the developmental stages that were studied. The spatial anisotropy and the temporal evolution across the developmental span in the textural attributes of embryonic fibronectin may have functional consequences for cell motility and morphogenesis. With this thesis, textural analysis has found yet another application, viz. the study of the surface structural attributes of an extracellular matrix protein network in the context of developmental biology. The work presented in this thesis is the first of its kind in applying texture analysis with gray scale co-occurrence matrix method to study the spatial distribution of fibronectin matrix within a given stage of embryonic development and the temporal textural changes as the embryonic development progresses from stage 5 through stage 9

Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia

<p>Abstract</p> <p>Background</p> <p>Diminished calcium (Ca²⁺) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.</p> <p>Methodology/Principal Findings</p> <p>VSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP₃R) and the SR Ca²⁺pumps (SERCA 2 and 3). Generally, a decrease in IP₃R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP₃R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca²⁺in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca²⁺sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca²⁺in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca²⁺in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca²⁺responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.</p> <p>Conclusions/Significance</p> <p>This work demonstrates that the previously-reported reduced Ca²⁺signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP₃R Ca²⁺channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.</p

Cardiac dysfunction in the diabetic rat: quantitative evaluation using high resolution magnetic resonance imaging

BACKGROUND: Diabetes is a major risk factor for cardiovascular disease. In particular, type 1 diabetes compromises the cardiac function of individuals at a relatively early age due to the protracted course of abnormal glucose homeostasis. The functional abnormalities of diabetic myocardium have been attributed to the pathological changes of diabetic cardiomyopathy. METHODS: In this study, we used high field magnetic resonance imaging (MRI) to evaluate the left ventricular functional characteristics of streptozotocin treated diabetic Sprague-Dawley rats (8 weeks disease duration) in comparison with age/sex matched controls. RESULTS: Our analyses of EKG gated cardiac MRI scans of the left ventricle showed a 28% decrease in the end-diastolic volume and 10% increase in the end-systolic volume of diabetic hearts compared to controls. Mean stroke volume and ejection fraction in diabetic rats were decreased (48% and 28%, respectively) compared to controls. Further, dV/dt changes were suggestive of phase sensitive differences in left ventricular kinetics across the cardiac cycle between diabetic and control rats. CONCLUSION: Thus, the MRI analyses of diabetic left ventricle suggest impairment of diastolic and systolic hemodynamics in this rat model of diabetic cardiomyopathy. Our studies also show that in vivo MRI could be used in the evaluation of cardiac dysfunction in this rat model of type 1 diabetes

Time-Dependent Alterations in Rat Macrovessels with Type 1 Diabetes

Vascular complications are associated with the progressive severity of diabetes, resulting in significant morbidity and mortality. This study quantifies functional vascular parameters and macrovascular structure in a rat model of type 1 diabetes. While there was no difference in the systemic arterial elastance (Ea) with 50 days of diabetes, changes were noted in the aorta and femoral artery including increased tunica media extracellular matrix content, decreased width of both the media and individual smooth muscle cell layers, and increased incidence of damaged mitochondria. Extracellular matrix proteins and elastin levels were significantly greater in the aorta of diabetic animals. These differences correlated with diminished matrix metalloprotease activity in the aorta of the diabetic animals. In conclusion, diabetes significantly altered the structure and ultrastructure of the aorta and femoral artery before systemic changes in arterial elastance could be detected

Spatial Anisotropies and Temporal Fluctuations in Extracellular Matrix Network Texture during Early Embryogenesis

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks — (a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis—as determined by fluctuating textural properties

The Role of Sleep in Motor Learning

Abstract Numerous factors affect motor learning, and its subsequent consolidation into effortlessly retrievable motor memories. Evidence from sleep research suggests a role for sleep in motor learning and motor memory consolidation. Optimal motor performance on a recently learned task is facilitated by sleepmediated consolidation of motor memories. Yet, sleep independent motor memory consolidation occurs for few tasks and do not appear to hamper performance. Growing evidence, however, is suggestive of a facilitative role for sleep in motor learning. Meanwhile the mechanisms underlying sleepmediated consolidation of motor memories are not clear, and the possible relevance of hippocampus to motor memory in the context of sleep-state awaits exploration

The heart in type 1 diabetes: Characterization of structure, function and exercise-induced benefits in diabetic cardiomyopathy

Dissertation (Ph.D.)--University of Kansas, Physical Therapy & Rehabilitation Sciences, 2007.Diabetes mellitus compromises the structure and function of the cardiovascular system. We have characterized the structural and functional abnormalities of the diabetic myocardium using streptozotocin-induced (generic) and autoimmune-intolerant (specific) rat models of type 1 diabetes. In addition, we have identified potential structural, functional, and molecular correlates of exercise-induced benefits in the diabetic myocardium. The experimental models demonstrated highly compromised structure and function of myocardium in the diabetic state. Using structural magnetic resonance imaging, we were able to demonstrate the abnormal heart wall dynamics resulting from myocardial stiffness; a characteristic of the fibrotic heart in diabetes. Furthermore, the diabetic left ventricle manifested cardiac cycle abnormalities detectable via functional magnetic resonance imaging. Systolic and diastolic left ventricular functions were compromised in the diabetic heart. Microscopically, increased accumulation of interstitial collagen and decreased distribution of mitochondria were identifiable as the cardinal features of the diabetic myocardium. Endurance training, however, attenuated the structural and functional defects of the diabetic heart. Training prevented the development of myocardial fibrosis and loss of viable mitochondria in the diabetic heart. Training also ameliorated the systolic and diastolic dysfunctions of the ventricular pump in diabetes. Moreover, training-induced benefits were evident at the molecular level as decreased expression of myocardial protein kinase c (β II isoform); a critical protein implicated in the pathogenesis of diabetic cardiomyopathy. In summary, these investigations demonstrate the vulnerability of the heart for failure and the efficacy of exercise in attenuating the major cardiac abnormalities in type 1 diabetes

Texture measures at each embryonic stage (HH5 through HH7), averaged over orientation and spatial scale.

<p>The overall trend (spatial anisotropy and temporal fluctuations) in the textural measures during stages 5 through 7 was evident upon averaging the Haralick feature values (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038266#pone-0038266-g002" target="_blank">figures 2</a> through <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038266#pone-0038266-g003" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038266#pone-0038266-g004" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038266#pone-0038266-g005" target="_blank">5</a>) over orientation (0°, 45°, 90° and135°) and scale (offsets 1 through 4 corresponding to each orientation). Values are mean ± S.D. Fibrillin-2 lateral network at stage 5 is shown in green, while lateral and medial fibronectin networks are shown in red and blue, respectively. Statistically significant differences (P<0.05) between ROIs were represented by “ * ”.</p

The relative textural differences between lateral and medial fibronectin networks during HH stage 6 of embryonic development.

<p>During HH stage 6 of embryonic development, the medial fibronectin network (b) demonstrated increased values of inertia across all offsets (c) compared with the lateral fibronectin network (a). The correlation values (d) in both medial and lateral networks demonstrated similar fall-off trends as a function of offset whereas a relative decrease in uniformity (e) and homogeneity (f) values marked the medial fibronectin distribution along the PS. The local entropy values of the medial network (h) were higher than the lateral network (g) as well. PS: Primitive Streak, Scale bar: 100 µm.</p