Extended regimen of a levonorgestrel/ethinyl estradiol transdermal delivery system: Predicted serum hormone levels using a population pharmacokinetic model.

OBJECTIVE: This study employed population pharmacokinetic (popPK) models to predict levonorgestrel (LNG) and ethinyl estradiol (EE) exposure after dosing with the transdermal contraceptive TWIRLA® (LNG/EE TDS) as a 12-week extended regimen in a healthy female population. METHODS: PopPK models were developed using data from a previously published phase 1, open-label, randomized clinical trial, ATI-CL14 (NCT01243580), in 36 healthy individuals. Models used cycle 2 data from 18 individuals who received the LNG/EE TDS, delivering LNG 120 µg/day and EE 30 µg/day, followed by a 1-week TDS-free period. Noncompartmental PK analyses were performed on simulated concentration-time profiles of 12 consecutive weeks of LNG/EE TDS use. RESULTS: The simulated concentration-time profiles and PK parameters for the simulated extended regimen indicated that predicted LNG and EE exposures at week 12 were similar to week 3 (predicted geometric mean EE area under the concentration-time curve from time 0 to 168 h [AUC0-168] on week 3 was 0.2% lower than week 12 and LNG AUC0-168 on week 3 was 0.9% lower than week 12), suggesting both were at steady state by week 3. Therefore, no notable accumulation beyond that at week 3 is predicted for LNG and EE following a 12-week extended regimen. The results are supported by the accumulation ratios based on maximum concentration and the area under the curve being similar at weeks 3 and 12 for LNG and EE. CONCLUSION: These results indicate that a 12-week extended LNG/EE regimen would provide similar systemic hormonal exposure as that seen by week 3 in a standard 28-day regimen, without further hormonal accumulation. The data support the safe use of a non-daily, low-dose hormonal contraceptive in an extended regimen but should be confirmed in a clinical PK study.

Substrate Stiffness Regulates Proinflammatory Mediator Production through TLR4 Activity in Macrophages.

Clinical data show that disease adversely affects tissue elasticity or stiffness. While macrophage activity plays a critical role in driving disease pathology, there are limited data available on the effects of tissue stiffness on macrophage activity. In this study, the effects of substrate stiffness on inflammatory mediator production by macrophages were investigated. Bone marrow-derived macrophages were grown on polyacrylamide gels that mimicked the stiffness of a variety of soft biological tissues. Overall, macrophages grown on soft substrates produced less proinflammatory mediators than macrophages grown on stiff substrates when the endotoxin LPS was added to media. In addition, the pathways involved in stiffness-regulated proinflammation were investigated. The TLR4 signaling pathway was examined by evaluating TLR4, p-NF-κB p65, MyD88, and p-IκBα expression as well as p-NF-κB p65 translocation. Expression and translocation of the various signaling molecules were higher in macrophages grown on stiff substrates than on soft substrates. Furthermore, TLR4 knockout experiments showed that TLR4 activity enhanced proinflammation on stiff substrates. In conclusion, these results suggest that proinflammatory mediator production initiated by TLR4 is mechanically regulated in macrophages.

Glutamate affects dendritic morphology of neurons grown on compliant substrates.

Brain stiffness changes in response to injury or disease. As a secondary consequence, glutamate is released from neurons and astroglia. Two types of glutamate receptors, N-methyl-d-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, sense mechanotransduction, leading to downstream signaling in neurons. Recently, our group reported that these two receptors affect dendrite morphology in hippocampal neurons grown on compliant substrates. Blocking receptor activity has distinct effects on dendrites, depending on whether neurons are grown on soft or stiff gels. In the current study, we examine whether exposure to glutamate itself alters stiffness-mediated changes to dendrites in hippocampal neurons. We find that glutamate augments changes seen when neurons are grown on soft gels of 300 or 600 Pa, but in contrast, glutamate attenuates changes seen when neurons are grown on stiff gels of 3,000 Pa. These results suggest that there is interplay between mechanosensing and glutamate receptor activation in determining dendrite morphology in neurons.

Lipid rafts direct macrophage motility in the tissue microenvironment.

Infiltrating leukocytes are exposed to a wide range of tissue elasticities. While we know the effects of substrate elasticity on acute inflammation via the study of neutrophil migration, we do not know its effects on leukocytes that direct chronic inflammatory events. Here, we studied morphology and motility of macrophages, the innate immune cells that orchestrate acute and chronic inflammation, on polyacrylamide hydrogels that mimicked a wide range of tissue elasticities. As expected, we found that macrophage spreading area increased as substrate elasticity increased. Unexpectedly, we found that morphology did not inversely correlate with motility. In fact, velocity of steady-state macrophages remained unaffected by substrate elasticity, while velocity of biologically stimulated macrophages was limited on stiff substrates. We also found that the lack of motility on stiff substrates was due to a lack of lipid rafts on the leading edge of the macrophages. This study implicates lipid rafts in the mechanosensory mechanism of innate immune cell infiltration.

Preparation of DNA-crosslinked polyacrylamide hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

The effects of substrate elastic modulus on neural precursor cell behavior.

The spinal cord has a limited capacity to self-repair. After injury, endogenous stem cells are activated and migrate, proliferate, and differentiate into glial cells. The absence of neuronal differentiation has been partly attributed to the interaction between the injured microenvironment and neural stem cells. In order to improve post-injury neuronal differentiation and/or maturation potential, cell-cell and cell-biochemical interactions have been investigated. However, little is known about the role of stem cell-matrix interactions on stem cell-mediated repair. Here, we specifically examined the effects of matrix elasticity on stem cell-mediated repair in the spinal cord, since spinal cord injury results in drastic changes in parenchyma elasticity and viscosity. Spinal cord-derived neural precursor cells (NPCs) were grown on bis-acrylamide substrates with various rigidities. NPC growth, proliferation, and differentiation were examined and optimal in the range of normal spinal cord elasticity. In conclusion, limitations in NPC growth, proliferation, and neuronal differentiation were encountered when substrate elasticity was not within normal spinal cord tissue elasticity ranges. These studies elucidate the effect injury mediated mechanical changes may have on tissue repair by stem cells. Furthermore, this information can be applied to the development of future neuroregenerative biomaterials for spinal cord repair.

Polyelectrolyte complex membranes for prevention of post-surgical adhesions in neurosurgery.

Adhesions are bands of tissue that can form after the surgery and bind together the surrounding tissue in the vicinity of the surgical site. In this work, polyelectrolyte complex (PEC)-based membranes were developed and investigated for their potential application as anti-adhesion barriers after neurosurgery. The adhesion and migration behavior of fibroblast and mixed neuronal cells were also investigated. Fibroblasts cells neither adhered nor migrated onto the PEC material after 5 days in vitro. Similar behaviors were observed for neurons and astrocytes cells. Swelling experiments of these membranes showed that the membranes are extremely hygroscopic and absorb significant amount of water. Membranes containing 70% alginate absorbed the highest amount of water (442 ± 24% of dry wet). PEC content was highest in 50% alginate containing membranes (87.1 ± 6.7%). The membrane's mechanical properties were attributed to combined contribution of water absorption and PEC content. Drug release profiles were investigated using albumin as a model drug. Membranes containing 70% alginate showed highest initial drug release rate followed by membranes containing 60 and 50% alginate. Membranes were stable and did not dissolve for 1 month in phosphate buffer and lysozyme solutions.

Fibroblast morphology on dynamic softening of hydrogels.

Despite cellular environments having dynamic characteristics, many laboratories utilized static polyacrylamide hydrogels to study the ECM-cell relationship. To attain a more in vivo like environment, we have developed a dynamic, DNA-crosslinked hydrogel (DNA gel). Through the controlled delivery of DNA, we can temporally decrease or increase gel stiffness while expanding or contracting the gel, respectively. These dual mechanical changes make DNA gels a cell-ECM model for studying dynamic mechano-regulated processes, such as wound healing. Here, we characterized DNA gels on a mechanical and cellular level. In contrast to our previous publication, in which we examined the increasing stiffness effects on fibroblast morphology, we examined the effects of decreased matrix stiffness on fibroblast morphology. In addition, we quantified the bulk and/or local stress and strain properties of dynamic gels. Gels generated about 0.5 Pa stress and about 6-11% strain upon softening to generate larger and more circular fibroblasts. These results complemented our previous study, where dynamic gels contracted upon stiffening to generate smaller and longer fibroblasts. In conclusion, we developed a biomaterial that increases and decreases in stiffness while contracting and expanding, respectively. We found that the dynamic deformation directionality of the matrix determined the fibroblast morphology and possibly influences function.

Mechanical Properties of DNA-Crosslinked Polyacrylamide Hydrogels with Increasing Crosslinker Density.

DNA-cross-linked polyacrylamide hydrogels (DNA gels) are dynamic mechanical substrates. The addition of DNA oligomers can either increase or decrease the crosslinker density to modulate mechanical properties. These DNA-responsive gels show promise as substrates for cell culture and tissue-engineering applications, since the gels allow time-dependent mechanical modulation. Previously, we reported that fibroblasts plated on DNA gels responded to modulation in elasticity via an increase or decrease in crosslinker density. To better characterize fibroblast mechanical signals, changes in stress and elastic modulus of DNA gels were measured over time as crosslinker density altered. In a previous study, we observed that as crosslinker density decreased, stress was generated, and elasticity changed over time; however, we had not evaluated stress and elastic modulus measurements of DNA gels as crosslinker density increased. Here, we completed this set of fibroblast studies by reporting stress and elastic modulus measurements over time as the crosslinker density increased. We found that the stress generated and the elastic modulus alterations were correlated. Hence, it seemed impossible to separate the effect of stress from the effect of modulus changes for fibroblasts plated on DNA gels. Yet, previous results and controls revealed that stress contributed to fibroblast behavior.

PSD-95 alters microtubule dynamics via an association with EB3.

Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology.

UEV-1 is an ubiquitin-conjugating enzyme variant that regulates glutamate receptor trafficking in C. elegans neurons.

The regulation of AMPA-type glutamate receptor (AMPAR) membrane trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the factors that mediate these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR trafficking in vivo. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in the accumulation of GLR-1 in elongated accretions in neuron cell bodies and along the ventral cord neurites. Mutants also have a corresponding behavioral defect--a decrease in spontaneous reversals in locomotion--consistent with diminished GLR-1 function. The localization of other synaptic proteins in uev-1-mutant interneurons appears normal, indicating that the GLR-1 trafficking defects are not due to gross deficiencies in synapse formation or overall protein trafficking. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants, and that receptors arrive at these endosomes independent of clathrin-mediated endocytosis. UEV-1 homologs in other species bind to the ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in most tissues. Nevertheless, ubc-13 mutants are similar in phenotype to uev-1 mutants, suggesting that the two proteins do work together to regulate GLR-1 trafficking. Our results suggest that UEV-1 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking.

Automated Sholl analysis of digitized neuronal morphology at multiple scales: Whole cell Sholl analysis versus Sholl analysis of arbor subregions.

The morphology of dendrites and the axon determines how a neuron processes and transmits information. Neurite morphology is frequently analyzed by Sholl analysis or by counting the total number of neurites and branch tips. However, the time and resources required to perform such analysis by hand is prohibitive for the processing of large data sets and introduces problems with data auditing and reproducibility. Furthermore, analyses performed by hand or using course-grained morphometric data extraction tools can obscure subtle differences in data sets because they do not store the data in a form that facilitates the application of multiple analytical tools. To address these shortcomings, we have developed a program (titled "Bonfire") to facilitate digitization of neurite morphology and subsequent Sholl analysis. Our program builds upon other available open-source morphological analysis tools by performing Sholl analysis on subregions of the neuritic arbor, enabling the detection of local level changes in dendrite and axon branching behavior. To validate this new tool, we applied Bonfire analysis to images of hippocampal neurons treated with 25 ng/ml brain-derived neurotrophic factor (BDNF) and untreated control neurons. Consistent with prior findings, conventional Sholl analysis revealed that global exposure to BDNF increases the number of neuritic intersections proximal to the soma. Bonfire analysis additionally uncovers that BDNF treatment affects both root processes and terminal processes with no effect on intermediate neurites. Taken together, our data suggest that global exposure of hippocampal neurons to BDNF results in a reorganization of neuritic segments within their arbors, but not necessarily a change in their number or length. These findings were only made possible by the neurite-specific Sholl data returned by Bonfire analysis.

Regulation of dendrite arborization by substrate stiffness is mediated by glutamate receptors.

Brain injury or disease can initiate changes in local or global stiffness of brain tissue. While stiffness of the extracellular environment is known to affect the morphology and function of many cell types, little is known about how the dendrites of neurons respond to changes in brain stiffness. To assess how extracellular stiffness affects dendrite morphology, we took biomaterial and biomedical engineering approaches. We cultured mixed and pure hippocampal neurons on hydrogels composed of polyacrylamide (PA) of varying stiffnesses to mimic the effects of extracellular matrix stiffness on dendrite morphology. The majority of investigations of cortical and spinal cord neurons on soft hydrogels examined branching at early time points (days in vitro (DIV) 2-7), an important distinction from our study, where we include later time points that encompass the peak of branching (DIV 10-12). At DIV 12, dendrite branching was altered by stiffness for both pure and mixed neuronal cultures. Furthermore, we treated hippocampal cultures with glutamate receptor antagonists and with astrocyte-conditioned media. Blocking AMPA and NMDA receptors affected the changes in dendrite branching seen at varying rigidities. Moreover, extracellular factors secreted by astrocytes also change dendrite branching seen at varying rigidities. Thus, astrocytes and ionotropic glutamate receptors contribute to mechanosensing.

Effects of substrate stiffness and cell density on primary hippocampal cultures.

Previous studies have shown that dendrites are influenced by substrate stiffness when neurons are plated in either pure or mixed cultures. However, because substrate rigidity can also affect other aspects of culture development known to impact dendrite branching, such as overall cell number, it is unclear whether substrate stiffness exerts a direct or indirect effect on dendrite morphology. In this study, we determine whether substrate stiffness plays a critical role in regulating dendrite branching independent of cell number. We plated primary mixed hippocampal cultures on soft and stiff gels, with Young's moduli of 1 kPa and 7 kPa, respectively. We found that neurons plated on stiffer substrates showed increased branching relative to neurons grown on softer substrates at the same cell number. On the stiff gels, we also observed a cell number-dependent effect, in which increasing initial plating density decreased dendrite branching. This change correlates with an increase in extracellular glutamate. We concluded that both cell number and substrate stiffness play roles in determining dendrite branching, and that the two effects are independent of one another.