Surgical treatment for subglottic stenosis often faces recurrence of narrowing and patients continue to face quality of life issues. Currently, biomechanical models that inform surgical treatments are difficult to create due to vocal fold intricacies and trouble distinguishing between tissue types-muscle, mucosa, and cartilage. Improved models of laryngeal microanatomy can be used to assess and predict surgical outcomes for patients who suffer from subglottic stenosis. While manual segmentation of histology is the standard, automatic segmentation can be more efficient, sensitive, and specific in tissue differentiation. Manual segmentation faces challenges like extensive operator time and inter-rater subjectivity. The aim of this study, therefore, was to qualitatively compare manual segmentation to automatic k-means segmentation of whole-slide histology rabbit vocal fold images. This is the preliminary step towards enhancing segmentation and, later, achieving a high-fidelity human larynx model.134 whole-slide histology images were manually segmented from seven rabbits using colored masks for muscle, cartilage, mucosa, and adipose tissue. Of those images, 31 were also selected for segmentation using k-means for initial qualitative comparison. At least two images were segmented per rabbit. K-means segmentation is optimal for our multidimensional data. Since it is deterministic, we selected the number of clusters as our parameter. After inspecting n=1-10 clusters, n=4 was chosen as qualitatively producing the most distinct clusters without oversampling.For our preliminary results, we compared the histological image, the manual segmentation, and the automatically segmented image. In the automatic segmentations, tissue type is distinguished by the grayscale contrast. The four classes visible are cartilage, muscle, adipose tissue, and mucosa. There is occasional loss of visualization of adipose tissue and mucosa, however, the cartilage and muscle tissues remain distinct. The mucosal area boundaries remain clear despite some visualization loss within the mucosal borders, therefore, mucosa can be distinguished from adjacent cartilage and muscle. Training on a large dataset like other machine learning algorithms is unnecessary for k-means, which is ideal for our relatively small sample size. The speed and versatility of k-means has qualitatively demonstrated comparable results to our manual segmentations.Future directions for our project include conducting k-means segmentation of the remainder of the 134 images collected. We have also tested mean-shift clustering and a deep learning convolutional neural network (CNN) to compare automatic segmentation methods to one another, and to the standard manual segmentation/ground truth. Future testing also includes quantitative analysis of these algorithms' accuracy. Beyond our histology images, we have at least 14 MR images (at least two per rabbit) of the larynges as well. We intend to combine and overlay the structural detail of the histology images with the soft tissue differentiation of the MRI.
Summary Clinical studies and experimental modeling identify a potential link between periodontal disease and periodontal pathogens such as P orphyromonas gingivalis and atherosclerosis and formation of macrophage foam cells. T oll‐like receptors and molecules governing their intracellular signaling pathways such as M y D 88 play roles in atherosclerosis, as well as host response to P . gingivalis . The aim of this study was to define roles of M y D 88 and T RIF during macrophage foam cell formation in response to P . gingivalis . In the presence of human low‐density lipoprotein ( LDL ) mouse bone‐marrow‐derived macrophages ( BM φ) cultured with P . gingivalis responded with significant reduction in tumor necrosis factor‐α ( TNF ‐α) and interleukin‐6 ( IL ‐6). The BM φ stained strongly with oil red O , regardless of whether bacterial challenge occurred concurrent with or before LDL treatment. Heat‐killed P . gingivalis stimulated foam cell formation in a similar way to live bacteria. The BM φ from M y D 88‐knockout and L ps2 mice revealed a significant role for M y D 88, and a minor role for T RIF in P . gingivalis ‐elicited foam cell formation. P orphyromonas gingivalis ‐elicited TNF ‐α and IL ‐6 were affected by M y D 88 ablation and to a lesser extent by T RIF status. These data indicate that LDL affects the TNF ‐α and IL ‐6 response of macrophages to P . gingivalis challenge and that M y D 88 and T RIF play important roles in P . gingivalis ‐elicited foam cell formation.
Abstract Background Brain aging is a major risk factor in the progression of cognitive diseases including Alzheimer’s disease (AD) and vascular dementia. We investigated a mouse model of brain aging up to 24 months old (mo). Methods A high field (11.7T) MRI protocol was developed to characterize specific features of brain aging including the presence of cerebral microbleeds (CMBs), morphology of grey and white matter, and tissue diffusion properties. Mice were selected from age categories of either young (3 mo), middle-aged (18 mo), or old (24 mo) and fed normal chow over the duration of the study. Mice were imaged in vivo with multimodal MRI, including conventional T2-weighted (T2W) and T2*-weighted (T2*W) imaging, followed by ex vivo diffusion-weighted imaging (DWI) and T2*W MR-microscopy to enhance the detection of microstructural features. Results Structural changes observed in the mouse brain with aging included reduced cortical grey matter volume and enlargement of the brain ventricles. A remarkable age-related change in the brains was the development of CMBs found starting at 18 mo and increasing in total volume at 24 mo, primarily in the thalamus. CMBs presence was confirmed with high resolution ex vivo MRI and histology. DWI detected further brain tissue changes in the aged mice including reduced fractional anisotropy, increased radial diffusion, increased mean diffusion, and changes in the white matter fibers visualized by color-coded tractography, including around a large cortical CMB. Conclusions The mouse is a valuable model of age-related vascular contributions to cognitive impairment and dementia (VCID). In composite, these methods and results reveal brain aging in older mice as a multifactorial process including CMBs and tissue diffusion alterations that can be well characterized by high field MRI.
Hypoxic-ischemic encephalopathy (HIE), resulting from a lack of blood flow and oxygen before or during newborn delivery, is a leading cause of cerebral palsy and neurological disability in children. Therapeutic hypothermia (TH), the current standard of care in HIE, is only beneficial in 1 of 7–8 cases. Therefore, there is a critical need for more efficient treatments. We have previously reported that omega-3 (n-3) fatty acids (FA) carried by triglyceride (TG) lipid emulsions provide neuroprotection after experimental hypoxic-ischemic (HI) injury in neonatal mice. Herein, we propose a novel acute therapeutic approach using an n-3 diglyceride (DG) lipid emulsions. Importantly, n-3 DG preparations had much smaller particle size compared to commercially available or lab-made n-3 TG emulsions. We showed that n-3 DG molecules have the advantage of incorporating at substantially higher levels than n-3 TG into an in vitro model of phospholipid membranes. We also observed that n-3 DG after parenteral administration in neonatal mice reaches the bloodstream more rapidly than n-3 TG. Using neonatal HI brain injury models in mice and rats, we found that n-3 DG emulsions provide superior neuroprotection than n-3 TG emulsions or TH in decreasing brain infarct size. Additionally, we found that n-3 DGs attenuate microgliosis and astrogliosis. Thus, n-3 DG emulsions are a superior, promising, and novel therapy for treating HIE.
Macrophages adapt both phenotypically and functionally to the cytokine balance in host tissue microenvironments. Recent studies established that macrophages contribute an important yet poorly understood role in the development of infection-elicited oral bone loss. We hypothesized that macrophage adaptation to inflammatory signals encountered before pathogen interaction would significantly influence the subsequent immune response of these cells to the keystone oral pathobiont Porphyromonas gingivalis. Employing classically activated (M1) and alternatively activated (M2) murine bone-marrow-derived macrophage (BMDMø), we observed that immunologic activation of macrophages before P. gingivalis challenge dictated phenotype-specific changes in the expression of inflammation-associated molecules important to sensing and tuning host response to bacterial infection including Toll-like receptors 2 and 4, CD14, CD18 and CD11b (together comprising CR3), major histocompatibility complex class II, CD80, and CD86. M2 cells responded to P. gingivalis with higher expression of tumor necrosis factor-α, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, regulated on activation normal T cell expressed and secreted, and KC than M1 cells. M1 BMDMø expressed higher levels of interleukin-10 to P. gingivalis than M2 BMDMø. Functionally, we observed that M2 BMDMø bound P. gingivalis more robustly than M1 BMDMø. These data describe an important contribution of macrophage skewing in the subsequent development of the cellular immune response to P. gingivalis.
Innate immune activation with expression of pro-inflammatory molecules such as TNF-α is a hallmark of the chronic inflammation associated with periodontal disease (PD). Porphyromonas gingivalis, a bacterium associated with PD, engages TLRs and activates MyD88-dependent and TIR-domain-containing adapter-inducing IFN-β (TRIF)-dependent signaling pathways. IFN regulatory factor (IRF) 3 is activated in a TRIF-dependent manner and participates in production of cytokines such as TNF-α; however, little is known regarding IRF3 and the host response to PD pathogens. We speculated that IRF3 participates in the host inflammatory response to P. gingivalis. Our results show that bone marrow macrophages (MØ) from WT mice respond to P. gingivalis with activation and nuclear translocation of IRF3. Compared with WT, MØ from IRF3 −/− , TRIF −/− , and TLR4 −/− mice responded with reduced levels of TNF-α on P. gingivalis challenge. In addition, full expression of IL-6 and RANTES by MØ to P. gingivalis was dependent on IRF3. Lastly, employing MØ from IRF3 −/− and IRF7 −/− mice we observed a significant role for IRF3 and a modest role for IRF7 in the P. gingivalis-elicited TNF-α response. These studies identify a role for IRF3 in the inflammatory response by MØ to the periodontal pathogen P. gingivalis.
Periodontal diseases are chronic oral inflammatory diseases that are polymicrobial in nature. The presence of specific bacteria in subgingival plaque such as Porphyromonas gingivalis is associated with microbial dysbiosis and the modulation of host immune response. Bacterially elicited innate immune activation and inflammation are key elements implicated in the destruction of soft and hard tissues supporting the teeth. Liver X receptors (LXRs) are nuclear hormone receptors with important function in lipid homeostasis, inflammation, and host response to infection; however, their contribution to chronic inflammatory diseases such as periodontal disease is not understood. The aim of this study was to define the contribution of LXRs in the development of immune response to P. gingivalis and to assess the roles that LXRs play in infection-elicited oral bone loss. Employing macrophages, we observed that P. gingivalis challenge led to reduced LXRα and LXRβ gene expression compared with that observed with unchallenged wild-type cells. Myeloid differentiation primary response gene 88 (MyD88)-independent, Toll/interleukin-1 receptor-domain-containing adapter-inducing interferon-β (TRIF)-dependent signaling affected P. gingivalis-mediated reduction in LXRα expression, whereas neither pathway influenced the P. gingivalis effect on LXRβ expression. Employing LXR agonist and mice deficient in LXRs, we observed functional effects of LXRs in the development of a P. gingivalis-elicited cytokine response at the level of the macrophage, and participation of LXRs in P. gingivalis-elicited oral bone loss. These findings identify novel importance for LXRs in the pathogenesis of P. gingivalis infection-elicited inflammation and oral bone loss.
Introduction: Omega-3 (n-3) fatty acids (FAs), specifically docohexaenoic acid (DHA) and eicosapentaenoic acid (EPA), act as bioactive unsaturated lipids with pleiotropic effects, affording neuroprotection in ischemic brain injury. Hypothesis: We reported that n-3 FAs injected acutely as triglyceride (TG) emulsions provide neuroprotection after ischemic brain injury. We now questioned whether novel lipid emulsions made from n-3 diglycerides (DG) would improve the delivery and effectiveness of n-3 FAs in brain after injury. Methods: We evaluated in vitro interactions of DG (DG-DHA) vs TG (TG-DHA) in phosphatidylcholine (PC) bilayer liposomes, as a model membrane system, by NMR spectroscopy. We compared the in vitro kinetics of DG vs TG hydrolysis by lipoprotein lipase. We investigated the neuroprotective effects of DG emulsions in a Vannucci murine model of hypoxic-ischemic (HI) brain injury. Results: NMR spectra of PC liposomes incubated with DG-DHA showed an additional peak, adjacent to the phospholipid carbonyl region, indicating a higher incorporation into PC bilayers and a narrower peak at almost the same position in a more fluid phase. In contrast, spectra of liposomes incubated with TG-DHA showed narrow peaks well-separated from PC resonances, representing phase-separated oil droplets. In lipolysis assays, DG emulsions had more efficient hydrolysis than TGs. Neonatal mice treated with DG-EPA, DG-DHA, or the combination of both (DG-DHA+EPA) after HI injury showed up to 3X better reduction in infarct volumes compared to TGs (p<0.05). Conclusions: Our data demonstrate that DG molecules incorporate in membrane bilayers more efficiently than TG. We postulate that the faster hydrolysis of DGs contributes to higher neuroprotection compared with TGs. Our findings indicate that n-3 DG emulsions represent a novel and much more efficient modality than n-3 TG for improving ischemic brain injury outcomes.
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