Characterization and validation of a spontaneous acute and protracted oxycodone withdrawal model in male and female mice.

Opioid use disorder (OUD) is a serious health problem that may lead to physical dependence, in addition to affective disorders. Preclinical models are essential for studying the neurobiology of and developing pharmacotherapies to treat these problems. Historically, chronic morphine injections have most often been used to produce opioid-dependent animals, and withdrawal signs indicative of dependence were precipitated by administering an opioid antagonist. In the present studies, we have developed and validated a model of dependence on oxycodone (a widely prescribed opioid) during spontaneous withdrawal in male and female C57BL/6J mice. Dependence was induced by chronically administering oxycodone through osmotic minipumps at different doses for 7 days. Somatic withdrawal signs were measured after 3, 6, 24, and 48 h following minipump removal. Additionally, sensitivity to mechanical, thermal, and cold stimuli, along with anxiety-like behavior, were also measured. Our results indicated that spontaneous withdrawal following discontinuation of oxycodone produced an increase in total withdrawal signs after 60 and 120 mg/kg/day regimens of oxycodone administration. These signs were reversed by the administration of clinically approved medications for OUD. In general, both female and male mice showed similar profiles of somatic signs of spontaneous withdrawal. Spontaneous withdrawal also resulted in mechanical and cold hypersensitivity lasting for 24 and 14 days, respectively, and produced anxiety-like behaviors after 2 and 3 weeks following oxycodone removal. These results help validate a new model of oxycodone dependence, including the temporally distinct emergence of somatic, hyperalgesic, and anxiety-like behaviors, potentially useful for mechanistic and translational studies of opioid dependence.

Histone H1.0 couples cellular mechanical behaviors to chromatin structure.

Tuning of genome structure and function is accomplished by chromatin-binding proteins, which determine the transcriptome and phenotype of the cell. Here we investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that histone H1.0, which compacts nucleosomes into higher-order chromatin fibers, controls genome organization and cellular stress response. We show that histone H1.0 has privileged expression in fibroblasts across tissue types and that its expression is necessary and sufficient to induce myofibroblast activation. Depletion of histone H1.0 prevents cytokine-induced fibroblast contraction, proliferation and migration via inhibition of a transcriptome comprising extracellular matrix, cytoskeletal and contractile genes, through a process that involves locus-specific H3K27 acetylation. Transient depletion of histone H1.0 in vivo prevents fibrosis in cardiac muscle. These findings identify an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling force generation, nuclear organization and gene transcription.

Incorporating Diversity, Equity, and Inclusion Content Into Bioengineering Curricula: A Program-Level Approach.

Diversity, equity, and inclusion (DEI) are interconnected with bioengineering, yet have historically been absent from accreditation standards and curricula. Toward educating DEI-competent bioengineers and meeting evolving accreditation requirements, we took a program-level approach to incorporate, catalog, and assess DEI content through the bioengineering undergraduate program. To support instructors in adding DEI content and inclusive pedagogy, our team developed a DEI planning worksheet and surveyed instructors pre- and post-course. Over the academic year, 74% of instructors provided a pre-term and/or post-term response. Of responding instructors, 91% described at least one DEI curricular content improvement, and 88% incorporated at least one new inclusive pedagogical approach. Based on the curricular adjustments reported by instructors, we grouped the bioengineering-related DEI content into five DEI competency categories: bioethics, inclusive design, inclusive scholarship, inclusive professionalism, and systemic inequality. To assess the DEI content incorporation, we employed direct assessment via course assignments, end-of-module student surveys, end-of-term course evaluations, and an end-of-year program review. When asked how much their experience in the program helped them develop specific DEI competencies, students reported a relatively high average of 3.79 (scale of 1 = "not at all" to 5 = "very much"). Additionally, based on student performance in course assignments and other student feedback, we found that instructors were able to effectively incorporate DEI content into a wide variety of courses. We offer this framework and lessons learned to be adopted by programs similarly motivated to train DEI-competent engineering professionals and provide an equitable, inclusive engineering education for all students.

Infarct Collagen Topography Regulates Fibroblast Fate via p38-Yes-Associated Protein Transcriptional Enhanced Associate Domain Signals.

RATIONALE: Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart's ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart. OBJECTIVE: To investigate the role of collagen organization in cardiac fibroblast fate determination. METHODS AND RESULTS: Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFß (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here. CONCLUSIONS: Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.

Regulators of cardiac fibroblast cell state.

Fibroblasts are the primary regulator of cardiac extracellular matrix (ECM). In response to disease stimuli cardiac fibroblasts undergo cell state transitions to a myofibroblast phenotype, which underlies the fibrotic response in the heart and other organs. Identifying regulators of fibroblast state transitions would inform which pathways could be therapeutically modulated to tactically control maladaptive extracellular matrix remodeling. Indeed, a deeper understanding of fibroblast cell state and plasticity is necessary for controlling its fate for therapeutic benefit. p38 mitogen activated protein kinase (MAPK), which is part of the noncanonical transforming growth factor ß (TGFß) pathway, is a central regulator of fibroblast to myofibroblast cell state transitions that is activated by chemical and mechanical stress signals. Fibroblast intrinsic signaling, local and global cardiac mechanics, and multicellular interactions individually and synergistically impact these state transitions and hence the ECM, which will be reviewed here in the context of cardiac fibrosis.

Guided vascularization in the rat heart leads to transient vessel patterning.

Recent progress in the production and maturation of iPSC-cardiomyocytes has facilitated major advances in building bioartificial heart tissue with functional cardiomyocytes. Despite this progress, vascularizing these constructs continues to be a barrier to clinical application. One emerging strategy for vascularization uses aligned "cords" of endothelial cells in tissue grafts to guide assembly of chimeric microvessels upon graft implantation. Here, we test whether this approach can guide vascularization of a bioartificial tissue implanted on the rat heart. We find that patterned cords of human endothelial cells anastomose and become perfused with host blood by 3 days post-implantation. Immunohistochemical staining confirmed that graft-derived micro-vessels persist in the patch for 7 days. Furthermore, we noted a shift in distribution of vessels in the patch from patterned cord-associated clustering at 3 days to a more diffuse distribution pattern at 7 days. This loss of patterning corresponded to an infiltration of CD68+ cells and an increase in collagen within the patch. Upon further engraftment of patches containing both cords and human cardiomyocytes, we identified human cardiomyocytes and graft derived vasculature at the time of explant. Our findings show that patterned endothelial cords guide transient vessel patterning on the rat heart. Our results also suggest that future work should be directed at further adapting vascularization strategies to the epicardial environment and add to an important emerging dialog in cardiac cell therapy that points to the need to characterize host response prior to or in parallel with efficacy studies.

A FRESH Take on Resolution in 3D Bioprinting.

Recent innovations in the materials used for bioprinting have enabled transformative gains in the resolution and architecture of 3D-printed engineered tissues. We focus here on one of these innovations, reported by Lee et al., which lowers the resolution limit for printing soft biomaterials.

Spatial presentation of biological molecules to cells by localized diffusive transfer.

Cellular decisions in human development, homeostasis, regeneration, and disease are coordinated in large part by signals that are spatially localized in tissues. These signals are often soluble, such that biomolecules produced by one cell diffuse to receiving cells. To recapitulate soluble factor patterning in vitro, several microscale strategies have been developed. However, these techniques often introduce new variables into cell culture experiments (e.g., fluid flow) or are limited in their ability to pattern diverse solutes in a user-defined manner. To address these challenges, we developed an adaptable method that facilitates spatial presentation of biomolecules across cells in traditional open cultures in vitro. This technique employs device inserts that are placed in standard culture wells, which support localized diffusive pattern transmission through microscale spaces between device features and adherent cells. Devices can be removed and cultures can be returned to standard media following patterning. We use this method to spatially control cell labeling with pattern features ranging in scale from several hundred microns to millimeters and with sequential application of multiple patterns. To better understand the method we investigate relationships between pattern fidelity, device geometry, and consumption and diffusion kinetics using finite element modeling. We then apply the method to spatially defining reporter cell heterogeneity by patterning a small molecule modulator of genetic recombination with the requisite sustained exposure. Finally, we demonstrate use of this method for patterning larger and more slowly diffusing particles by creating focal sites of gene delivery and infection with adenoviral, lentiviral, and Zika virus particles. Thus, our method leverages devices that interface with standard culture vessels to pattern diverse diffusible factors, geometries, exposure dynamics, and recipient cell types, making it well poised for adoption by researchers across various fields of biological research.

Extension and Application of High-Speed Digital Imaging Analysis Via Spatiotemporal Correlation and Eigenmode Analysis of Vocal Fold Vibration Before and After Polyp Excision.

OBJECTIVES/HYPOTHESIS: To evaluate the spatiotemporal correlation of vocal fold vibration using eigenmode analysis before and after polyp removal and explore the potential clinical relevance of spatiotemporal analysis of correlation length and entropy as quantitative voice parameters. We hypothesized that increased order in the vibrating signal after surgical intervention would decrease the eigenmode-based entropy and increase correlation length. STUDY DESIGN: Prospective case series. METHODS: Forty subjects (23 males, 17 females) with unilateral (n = 24) or bilateral (n = 16) polyps underwent polyp removal. High-speed videoendoscopy was performed preoperatively and 2 weeks postoperatively. Spatiotemporal analysis was performed to determine entropy, quantification of signal disorder, correlation length, size, and spatially ordered structure of vocal fold vibration in comparison to full spatial consistency. The signal analyzed consists of the vibratory pattern in space and time derived from the high-speed video glottal area contour. RESULTS: Entropy decreased (Z = -3.871, P < .001) and correlation length increased (t = -8.913, P < .001) following polyp excision. The intraclass correlation coefficients (ICC) for correlation length and entropy were 0.84 and 0.93. CONCLUSION: Correlation length and entropy are sensitive to mass lesions. These parameters could potentially be used to augment subjective visualization after polyp excision when evaluating procedural efficacy.

Expression of Surfactant Protein-A during LPS-Induced Otitis Media with Effusion in Mice.

OBJECTIVE: The objective of this study was to investigate the expression and role of surfactant protein (SP) in the middle ear throughout lipopolysaccharide (LPS)-induced otitis media with effusion (OME). STUDY DESIGN: Randomized case-controlled animal model. SETTING: Shandong University, Jinan, China. SUBJECTS AND METHODS: SP expression was monitored using reverse transcription polymerase chain reaction (PCR) in normal mice (n = 5). Two groups, control phosphate-buffered saline-injected mice (n = 5) and LPS-injected mice (n = 5), were euthanized 5 days following injection. RNA was extracted for reverse transcription PCR and real-time PCR, and temporal bone samples were used for hematoxylin and eosin staining. LPS was injected into mice, and 5 mice per test were euthanized at 0, 12, 24, 48, 72, and 96 hours following injection. For mRNA expression quantification, reverse transcription PCR and real-time PCR were performed, and proinflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: SP-A and SP-D expression was detected in normal murine Eustachian tubes. SP-A expression was up-regulated after LPS-induced OME (P = .01). At various time points after LPS injection, concentrations of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin (IL)-1ß, and IL-6) in the middle ear increased significantly (P < .05) and correlated with changes in SP-A expression. CONCLUSION: SP-A and SP-D exist in the murine middle ear. The expression of SP-A and TNF-α, IL-1ß, and IL-6 was up-regulated in the middle ear of the LPS-induced OME animal model.

Influence of intraocular astigmatism on the correction of myopic astigmatism by femtosecond laser small-incision lenticule extraction.

PURPOSE: To evaluate the influence of the origin of astigmatism on the correction of myopia or myopic astigmatism by femtosecond laser small-incision lenticule extraction (SMILE). SETTING: Ophthalmology Department, Eye and ENT Hospital, Shanghai, China. DESIGN: Prospective case series. METHODS: Small-incision lenticule extraction was performed to correct myopia or myopic astigmatism. Ocular residual astigmatism (ORA) was determined by vector analysis using manifest refraction and Scheimpflug camera imaging of the anterior cornea. Patients were divided into 2 groups according to ORA (high >1.0 diopter [D]; low ≤1.0 D), and procedural efficacy was compared. Patients were examined preoperatively and 1, 3, and 6 months postoperatively. RESULTS: This study comprised 122 right eyes of 122 patients. No significant difference was found in the preoperative manifest astigmatism (target-induced astigmatism [TIA]) between the low ORA group (n = 67) and high ORA group (n = 55). The mean postoperative manifest astigmatism was higher in the high ORA group at all postoperative timepoints (1 month: t = 2.182, P=.031; 3 months: t = 2.30, P=.023; 6 months: t = 2.193, P=.03). The mean index of success (postoperative astigmatism/TIA) was 0.68 in the high ORA group and 0.34 in the low ORA group 1 month postoperatively (t = 2.531, P=.013); 0.73 and 0.39, respectively, at 3 months (t = 2.689, P=.008); and 0.77 and 0.46, respectively, at 6 months (t = 2.105, P=.037). CONCLUSION: Small-incision lenticule extraction was effective in correcting astigmatism but may be less effective in correcting ORA.

Influence of intraocular astigmatism on the correction of myopic astigmatism by laser-assisted subepithelial keratectomy.

PURPOSE: To assess the influence of the origin of astigmatism on the correction of myopic astigmatism by laser-assisted subepithelial keratectomy (LASEK). SETTING: Ophthalmology Department, Eye and ENT Hospital, Shanghai, China. DESIGN: Prospective study. METHODS: Patients having LASEK to correct myopia or myopic astigmatism were divided into 2 groups according to their ocular residual astigmatism (ORA). Patients were examined preoperatively and 1 and 3 months postoperatively. The efficacy of LASEK was compared between those with and those without a significant amount of intraocular astigmatism. RESULTS: The study comprised 54 eyes of 54 patients. The mean index of success (ratio of magnitude of remaining uncorrected astigmatism to that of initial preoperative astigmatism) in the high ORA group (n=21) and low ORA group (n=33) was 0.85 and 0.48, respectively, 1 month after surgery (t=2.17, P=.04) and 0.88 and 0.32, respectively, 3 months after surgery (t=2.18, P=.04). The Zernike coefficient of horizontal coma, Z(3,+1), increased more after surgery in the high ORA group than in the low ORA group (1 month versus preoperative, t=2.32, P=.024; 3 months versus preoperative, t=2.07, P=.048). CONCLUSIONS: Nine percent and 2% of the eyes had minimal corneal haze at 1 month and 3 months, respectively. Laser-assisted subepithelial keratectomy was less effective in correcting myopic astigmatism when astigmatism was mainly located at the internal optics. Horizontal coma increased more after LASEK in patients with higher ORA. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.