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Biomedical subjects

D G Simpson

Publications and source records attributed to D G Simpson.

At least 19 recordsLinked to original sources
PubMed2005

Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning.

We characterize the infiltration of interstitial cells into tissue engineering scaffolds prepared with electrospun collagen, electrospun gelatin, electrospun poly(glycolic) acid (PGA), electrospun poly(lactic) acid (PLA), and an electrospun PGA/PLA co-polymer. Electrospinning conditions were optimized to produce non-woven tissue engineering scaffolds composed of individual fibrils less than 1000 nm in diameter. Each of these materials was then electrospun into a cylindrical construct with a 2 mm inside diameter with a wall thickness of 200-250 microm. Electrospun scaffolds of collagen were rapidly, and densely, infiltrated by interstitial and endothelial cells when implanted into the interstitial space of the rat vastus lateralis muscle. Functional blood vessels were evident within 7 days. In contrast, implants composed of electrospun gelatin or the bio-resorbable synthetic polymers were not infiltrated to any great extent and induced fibrosis. Our data suggests that topographical features, unique to the electrospun collagen fibril, promote cell migration and capillary formation.

Animals↗
PubMed2001

Superthreshold behavior and threshold estimation of ultrasound-induced lung hemorrhage in adult mice and rats.

Threshold estimates and superthreshold behaviors for ultrasound-induced lung hemorrhage were investigated as a function of species (adult mice and rats) and ultrasound frequency (2.8 and 5.6 MHz). A total of 151 6-to-7-week-old female ICR mice and 160 10-to-11-week-old female Sprague-Dawley rats were randomly divided into two ultrasonic frequency groups, and further randomly divided into seven or eight ultrasonic peak rarefactional pressure groups. Each group consisted of about 10 animals. Animals were exposed to pulsed ultrasound at either 2.8-MHz center frequency (1-kHz PRF, 1.42-microsecond pulse duration) or 5.6-MHz center frequency (1-kHz PRF, 1.17-microsecond pulse duration) for a duration of 10 seconds. The in situ (at the pleural surface) peak rarefactional pressure levels ranged between 2.5 and 10.5 MPa for mice and between 2.3 and 11.3 MPa for rats. The mechanical index (MI) ranged between 1.4 and 6.3 at 2.8 MHz for mice and between 1.1 and 3.1 at 5.6 MHz for rats. The lesion surface area and depth were measured for each animal as well as the percentage of animals with lesions per group. The characteristics of the lesions produced in mice and rats were similar to those described in previous studies by our research group and others, suggesting a common pathogenesis in the initiation and propagation of the lesions at the gross and microscopic levels. The percentage of animals with lesions showed no statistical differences between species or between ultrasound frequencies. These findings suggest that mice and rats are similar in sensitivity to ultrasound-induced lung damage and that the occurrence of lung damage is independent of frequency. Lesion depth and surface area also showed no statistically significant differences between ultrasound frequencies for mice and rats. However, there was a significant difference between species for lesion area and a suggestive difference between species for lesion depth. The superthreshold behavior of lesion area and depth showed that rat lung had more damage than mouse lung, and the threshold estimates showed a weak, or lack of, frequency dependency, suggesting that the MI is not consistent with the observed findings.

Animals↗
PubMed2001

Superthreshold behavior and threshold estimates of ultrasound-induced lung hemorrhage in adult rats: role of beamwidth.

It is well documented that ultrasound-induced lung hemorrhage can occur in mice, rats, rabbits, pigs, and monkeys. The objective of this study was to assess the role of the ultrasound beamwidth (beam diameter incident on the lung surface) on lesion threshold and size. A total of 144 rats were randomly exposed to pulsed ultrasound at three exposure levels and four beamwidths (12 rats per group). The three in situ peak rarefactional pressures were about 5, 7.5, and 10 MPa. The four 19-mm-diameter focused transducers had measured pulse-echo -6-dB focal beamwidths of 470 microm (2.8 MHz; f/1), 930 microm (2.8 MHz; f/2), 310 microm (5.6 MHz; f/1), and 510 microm (5.6 MHz; f/2). Exposure durations were 10 s, pulse repetition frequencies were 1 kHz, and pulse durations were 1.3 micros (2.8 MHz; f/1), 1.2 micros (2.8 MHz; f/2), 0.8 micros (5.6 MHz; f/1) and 1.1 micros (5.6 MHz; f/2). The lesion surface area and depth were measured for each rat as well as the percentage of rats with lesions per group. Logistic regression analysis and Gaussian-Tobit analysis methods were used to analyze the data. The effects of in situ peak rarefactional pressure and beamwidth were highly significant, but ultrasonic frequency was not significant. In addition, the estimated interaction between in situ peak rarefactional pressure and beamwidth was positive and highly significant. The ultrasound beamwidth incident on the lung surface was shown to strongly affect the percentage and size of ultrasound-induced lung hemorrhage lesions. Even though ultrasonic frequency was an experimental variable, it was not shown to affect the lesion percentage or size.

Animals↗
PubMed2000

Random effects in censored ordinal regression: latent structure and Bayesian approach.

This paper discusses random effects in censored ordinal regression and presents a Gibbs sampling approach to fit the regression model. A latent structure and its corresponding Bayesian formulation are introduced to effectively deal with heterogeneous and censored ordinal observations. This work is motivated by the need to analyze interval-censored ordinal data from multiple studies in toxicological risk assessment. Application of our methodology to the data offers further support to the conclusions developed earlier using GEE methods yet provides additional insight into the uncertainty levels of the risk estimates.

Animals↗
PubMed1999

Regulation of cardiac myocyte protein turnover and myofibrillar structure in vitro by specific directions of stretch.

We have examined how different degrees (0.5%, 1.0%, 2.5%, 5.0%, and 10.0%) and directions of stretch regulate the turnover and accumulation of contractile proteins in cultured neonatal cardiac myocytes (NCMs). In pulse-chase experiments, stellate-shaped NCMs with random arrays of myofibrils (MFs) exhibited a threshold response to stretch. With respect to unstretched controls, the turnover of the contractile protein pool was suppressed 50% to 100% in stellate NCMs stretched 1.0% to 5.0% and was unaltered in stellate NCMs stretched 0.5% or 10.0%. The posttranslational metabolism of myosin heavy chain (MHC) and actin was regulated in parallel with the total contractile protein pool. The turnover of the cytoplasmic protein pool remained unchanged in response to stretch. NCMs plated onto an aligned matrix of type I collagen expressed an elongated, rod-like cell shape. The MFs of these cells were distributed in parallel with one another along a single unique axis. The tissue-like pattern of organization of these cultures made it possible to assay how specific directions of stretch affected cardiac protein turnover and MF organization. In pulse-chase experiments, stretch in parallel with the MFs did not alter the turnover of the total contractile protein pool, the cytoplasmic protein pool, MHC, or actin. The total cellular concentration of MHC and actin remained constant, and MF alignment was not overtly affected. In contrast, even modest degrees of stretch across the short axis of the MFs suppressed total contractile protein turnover, the turnover of MHC and actin, and promoted the accumulation of these MF subunits. The parallel alignment of MFs deteriorated in myocytes stretched greater than 5%. The characteristic response of aligned myocytes to stretch was not affected by the contractile state of the cells. Isoproterenol (ISO) treatment in concert with stretch in parallel with the MFs modestly accelerated contractile protein turnover. Conversely, contractile protein turnover was suppressed in cells treated with ISO and stretched across the short axis of the MFs. Contractile arrest with nifedipine (NIFED) accelerated total myofibrillar protein turnover. Stretch across the short axis, but not in parallel with the MFs, suppressed protein turnover in cells treated with NIFED. The turnover of the cytosolic proteins remained constant under all conditions assayed. These data suggest that specific directions of stretch may play a crucial role in regulating MF organization and the metabolism of contractile proteins in the cardiac myocyte.

Actins↗
PubMed1999

Regression modeling of ordinal data with nonzero baselines.

This paper develops regression models for ordinal data with nonzero control response probabilities. The models are especially useful in dose-response studies where the spontaneous or natural response rate is nonnegligible and the dosage is logarithmic. These models generalize Abbott's formula, which has been commonly used to model binary data with nonzero background observations. We describe a biologically plausible latent structure and develop an EM algorithm for fitting the models. The EM algorithm can be implemented using standard software for ordinal regression. A toxicology data set where the proposed model fits the data but a more conventional model fails is used to illustrate the methodology.

Algorithms↗
PubMed1998

Steady state visual evoked potential (SSVEP) changes in response to olfactory stimulation.

The location, nature and characteristics of brain activity during detection and identification of odors are of importance if brain function techniques are to be of value to sensory systems. Steady state probe topography (SSPT) has been used in our laboratory to record steady state visual evoked potentials (SSVEP), which have demonstrated cognitive task-related changes in a variety of testing paradigms. The enhanced spatial (when using 64 channels) and temporal resolution (380 ms) of the system enable the brain electrical activity changes occurring before, during and after delivery of an odor to be examined. We have developed a system which can deliver odors during normal respiration and the accurate timing needed for SSVEP recordings. The system is based on the premise that a subject breathing butanol compared with filtered medical air will demonstrate SSVEP topographic changes associated with detection and identification of butanol. During our experiments the subject has either an air sample or an equal volume of butanol injected into the inspiratory airflow. These are randomized, and every breath has the same stimulus system with no known clues as to the differences apart from detection. The results from a panel of 10 female subjects--who all identified the butanol correctly--showed that butanol delivery resulted in sequences of changes in SSVEP topography (amplitude and latencies) which involved parietal, frontal and temporal regions. While consistent with other studies (parietal changes), our results revealed more dynamic temporal changes involving prefrontal and parietal regions at different periods around odor delivery.

Brain↗
PubMed1997

The effects of angiotensin II and specific angiotensin receptor blockers on embryonic cardiac development and looping patterns.

The role of angiotensin II (Ang II) in the early embryonic development of the heart has not been examined. We have used RT-PCR to identify mRNA for angiotensinogen, angiotensin-converting enzyme, and the Ang II AT1 and AT2 receptors in embryonic day 10.25 Sprague-Dawley rats, and have used confocal microscopy to localize the AT1 receptor to the greater curvature of the developing ventricle in these animals at embryonic days (ED) 9.25 and 10.25. The antibodies used in immunolocalization studies did not distinguish between the AT1a and AT1b receptor subtypes. In whole embryo culture, Ang II added to the culture media resulted in increased ventricular growth and myocyte hypertrophy when treated embryos were compared to cultured littermate controls. Use of Losartan and PD123,319 to block the Ang II AT1 and AT2 receptors resulted in reduced ventricular development and cardiac dilation when compared to control and Ang II-treated embryos. Addition of Ang II and PD123,319 to the culture media also resulted in cardiac loop inversions which may be associated with disruption of normal myofibrillar development. These results clearly indicate an important role for Ang II in the early embryonic development of the heart.

Actins↗
PubMed1997

Analysis of heart development in cultured rat embryos.

The long-range goal of this research is to establish an in vitro system that will permit pertubation of mammalian heart development and in situ examination of the cellular and molecular events underlying cardiac morphogenesis. Rat embryos at 9.5-11.5 days of gestation were placed in culture bottles containing rat serum and Tyrode's solution. Embryos cultured for 24 and 48 h were compared to age-matched in vivo controls for morphological score, morphometric analysis of heart development, and confocal and electron microscopic analysis of myofiber pattern formation. Morphological scores indicated that embryos cultured for 24 h from day 9.5 to 10.5 had essentially normal development when compared to age-matched embryos allowed to develop in vivo. Development of embryos maintained for 48 h in culture was slightly delayed at 66-68% of age matched in vivo embryos. Analysis of hearts from embryos allowed to develop 9.5-11.5 days in vivo plus 24 and 48 h in culture showed that the ventricular thickness and height, as well as the truncal, atrial and ventricular diameters were equivalent to those of hearts from age-matched in vivo controls. Hearts from embryos allowed to develop from 11.5-12.5 days in vitro and cultured for 24 and 48 h had smaller left ventricular and atrial dimensions than controls. Cardiac myofibrillogenesis and myofibrillar pattern formation in embryos cultured from 9.5 days of in vivo development for 48 h were also normal. These studies indicate that the rat whole embryo culture system is a useful model to study several critical periods in mammalian heart development.

Animals↗
PubMed1997

Vinculin is an essential component for normal myofibrillar arrangement in fetal mouse cardiac myocytes.

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5+/-26.8% and 48.7+/-20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2+/-14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.

Animals↗
PubMed1997

Categorical regression analysis of acute exposure to tetrachloroethylene.

Exposure-response analysis of acute noncancer risks should consider both concentration (C) and duration (T) of exposure, as well as severity of response. Stratified categorical regression is a form of meta-analysis that addresses these needs by combining studies and analyzing response data expressed as ordinal severity categories. A generalized linear model for ordinal data was used to estimate the probability of response associated with exposure and severity category. Stratification of the regression model addresses systematic differences among studies by allowing one or more model parameters to vary across strata defined, for example, by species and sex. The ability to treat partial information addresses the difficulties in assigning consistent severity scores. Studies containing information on acute effects of tetrachloroethylene in rats, mice, and humans were analyzed. The mouse data were highly uncertain due to lack of data on effects of low concentrations and were excluded from the analysis. A model with species-specific concentration intercept terms for rat and human central nervous system data improved fit to the data compared with the base model (combined species). More complex models with strata defined by sex and species did not improve the fit. The stratified regression model allows human effect levels to be identified more confidently by basing the intercept on human data and the slope parameters on the combined data (on a C x T plot). This analysis provides an exposure-response function for acute exposures to tetrachloroethylene using categorical regression analysis.

Administration, Inhalation↗
PubMed1997

Mechanical forces regulate focal adhesion and costamere assembly in cardiac myocytes.

To determine whether the formation and maintenance of focal adhesions and costameres in cardiac myocytes are influenced by the mechanical forces that they transmit, we mechanically unloaded these cells by inhibiting their spontaneous contractile activity with the calcium-channel blocker nifedipine (12 microM). Interference-reflection and fluorescence microscopy revealed that within 24 h of arrest, beta 1-integrin- and vinculin-positive focal adhesions and costameres were disrupted. Loss of mature beta 1-integrin from the cell surface was observed in cell surface-labeling experiments and in Western blots. Subjecting nonbeating cells to a 5% static stretch for 24 h resulted in an increase of 21% for beta 1-integrin and 39% for vinculin. Stretching beating cells resulted in 71 and 9% increases, respectively. Intracellular concentrations of pre-beta 1 were not affected by contractile activity or by stretch. Our results indicate that mechanical forces stabilize the cellular levels of beta 1-integrin and vinculin by possibly regulating their association with the formation and maintenance of focal adhesions and costameres.

Animals↗
PubMed1996

Mechanical regulation of cardiac myocyte protein turnover and myofibrillar structure.

Mechanical forces play an essential role in regulating the synthesis and assembly of contractile proteins into the sarcomeres of cardiac myocytes. To examine if physical forces might also regulate the turnover of contractile proteins at a posttranslational site of control, beating and nonbeating neonatal cardiac myocytes (NCM) were subjected to a 5% static stretch. The L-type calcium channel blocker nifedipine (12 microM) was used to inhibit contraction. Pulse-chase biosynthetic labeling experiments demonstrated that contractile arrest accelerated the loss of isotopic tracer from the total myofibrillar protein fraction, myosin heavy chain (MHC), and actin, but not desmin. Myofibrillar abnormalities developed in parallel with these metabolic changes. A 5% static load appeared to partially stabilize myofibrillar structure in nonbeating NCM and suppressed the loss of isotopic tracer from the total myofibrillar protein fraction, MHC, and actin in beating and nonbeating NCM. Contractile activity and/or a static stretch promoted the accumulation of MHC, actin, and desmin. Applying a static load to myocytes that lacked preexisting myofibrils did not promote the assembly of sarcomeres or alter protein turnover. These data indicate that the turnover of MHC and actin is correlated with the organizational state of the myofibrillar apparatus.

Animals↗
PubMed1995

Mechanical regulation of cardiac myofibrillar structure.

The excitation-contraction coupling cycle (ECC) consists of a complex cascade of electrochemical and mechanical events; however, the relative contributions of these different processes in the regulation of cardiac myofibrillar structure are not well understood. There is extensive evidence to suggest that the mechanical aspects of the ECC play a crucial role in controlling the availability of contractile proteins for myofibrillar assembly. To examine if these physical forces might also serve to stabilize the structure of preexisting myofibrils, beating and nonbeating cultures of neonatal cardiac myocytes (NCM) were subjected to a 5% static stretch. Contractile arrest was achieved by treating NCM with 12 microM nifedipine, which resulted in immediate and sustained contractile arrest and initiated the evolution of marked myofibrillar abnormalities within 24 hours. As judged by scanning confocal and transmission electron microscopic examination, an external load appears to partially stabilize myofibrillar structure in nonbeating NCM. These results suggest that the maintenance of myofibrillar structure may be highly dependent upon the mechanical aspects of ECC.

Animals↗
PubMed1994

Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

Animals↗
PubMed1993

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes.

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non-beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.

Actins↗