Organization Of Fibers Research Articles

A novel framework for in-vivo diffusion tensor distribution MRI of the human brain

Neural tissue microstructure plays an important role in developmental, physiological and pathophysiological processes. Diffusion tensor distribution (DTD) MRI helps probe subvoxel heterogeneity by describing water diffusion within a voxel using an ensemble of non-exchanging compartments characterized by a probability density function of diffusion tensors. In this study, we provide a new framework for acquiring multiple diffusion encoding (MDE) images and estimating DTD from them in the human brain in vivo. We interfused pulsed field gradients (iPFG) in a single spin echo to generate arbitrary b-tensors of rank one, two, or three without introducing concomitant gradient artifacts. Employing well-defined diffusion encoding parameters we show that iPFG retains salient features of a traditional multiple-PFG (mPFG/MDE) sequence while reducing the echo time and coherence pathway artifacts thereby extending its applications beyond DTD MRI. Our DTD is a maximum entropy tensor-variate normal distribution whose tensor random variables are constrained to be positive definite to ensure their physicality. In each voxel, the second-order mean and fourth-order covariance tensors of the DTD are estimated using a Monte Carlo method that synthesizes micro-diffusion tensors with corresponding size, shape, and orientation distributions to best fit the measured MDE images. From these tensors we obtain the spectrum of diffusion tensor ellipsoid sizes and shapes, and the microscopic orientation distribution function (μODF) and microscopic fractional anisotropy (μFA) that disentangle the underlying heterogeneity within a voxel. Using the DTD-derived μODF, we introduce a new method to perform fiber tractography capable of resolving complex fiber configurations. The results revealed microscopic anisotropy in various gray and white matter regions and skewed MD distributions in cerebellar gray matter not observed previously. DTD MRI tractography captured complex white matter fiber organization consistent with known anatomy. DTD MRI also resolved some degeneracies associated with diffusion tensor imaging (DTI) and elucidated the source of diffusion heterogeneity which may help improve the diagnosis of various neurological diseases and disorders.

Behavioral correlates of fascicular organization: The confluence of muscle architectural anatomy and function.

Muscle is a complex tissue that has been studied on numerous hierarchical levels: from gross descriptions of muscle organization to cellular analyses of fiber profiles. In the middle of this space between organismal and cellular biology lies muscle architecture, the level at which functional correlations between a muscle's internal fiber organization and contractile abilities are explored. In this review, we summarize this relationship, detail recent advances in our understanding of this form-function paradigm, and highlight the role played by The Anatomical Record in advancing our understanding of functional morphology within muscle over the past two decades. In so doing, we honor the legacy of Editor-in-Chief Kurt Albertine, whose stewardship of the journal from 2006 through 2020 oversaw the flourishing of myological research, including numerous special issues dedicated to exploring the behavioral correlates of myology across diverse taxa. This legacy has seen the The Anatomical Record establish itself as a preeminent source of myological research, and a true leader within the field of comparative anatomy and functional morphology.

Effect of cold atmospheric plasma/NO gas application with different exposure times on healing in wounds with tissue loss in diabetic rats.

Applications of cold atmospheric plasma/nitric oxide (CAP/NO) gas have recently garnered popularity when treating impaired wound healing in patients with diabetes. In this study, we aimed to investigate the effects of NO gas application for 60 and 120 s on wound healing in diabetic rats. A dorsal excision 3 cm in diameter was performed in 15 diabetic rats; these rats were categorized into the following 3 groups: DC (untreated diabetic control); DNO/60 (exposure to 200 ppm NO gas for 60 s/day); and DNO/120 (exposure to 200 ppm NO gas for 120 s/day). Wound contraction on days 0, 3, 7, 11, and 14 and wound contraction rate between days 0 and 14 were evaluated. On day 14, tissue samples were collected for histopathologic assessment of inflammation, epithelial regeneration, angiogenesis congestion, and collagen fiber organization. Normality of distribution was assessed using the Shapiro-Wilk test, and intergroup comparisons were performed using the Mann-Whitney U test (NPar Test) and the Kruskal-Wallis test (non-parametric ANOVA). Wound contraction during treatment days 7-14 was significantly greater in the NO-treatment groups than in the DC group (p<0.05). The NO60 s and NO120 s groups showed a significantly higher wound contraction rate than the DC group (p=0.033, p=0.049, respectively). Significant differences were noted between the control and NO groups in terms of inflammation (p<0.05) and between the control group and DNO/60 and DNO/120 groups in terms of collagen organization (p<0.05, p<0.01, respectively). Evaluation of epithelialization revealed significant intergroup differences between the control and NO treatment groups (p<0.01). In this study, the application of NO once a day for 60 seconds and 120 seconds in diabetic wounds contributed equally to wound healing.

Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants.

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including µCT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRµCT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2 -/- mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2 -/- mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2 -/- zebrafish, and demonstrate the promise of DICE-PPC-SRµCT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.

Going nuclear: Molecular adaptations to exercise mediated by myonuclei.

Muscle fibers are multinucleated, and muscle fiber nuclei (myonuclei) are believed to be post-mitotic and are typically situated near the periphery of the myofiber. Due to the unique organization of muscle fibers and their nuclei, the cellular and molecular mechanisms regulating myofiber homeostasis in unstressed and stressed conditions (e.g., exercise) are unique. A key role myonuclei play in regulating muscle during exercise is gene transcription. Only recently have investigators had the capability to identify molecular changes at high resolution exclusively in myonuclei in response to perturbations in vivo. The purpose of this review is to describe how myonuclei modulate their transcriptome, epigenetic status, mobility and shape, and microRNA expression in response to exercise in vivo. Given the relative paucity of high-fidelity information on myonucleus-specific contributions to exercise adaptation, we identify specific gaps in knowledge and provide perspectives on future directions of research.

Removal of GAGs Regulates Mechanical Properties, Collagen Fiber Formation, and Alignment in Tissue Engineered Meniscus.

The complex fibrillar architecture of native meniscus is essential for proper function and difficult to recapitulate in vitro. In the native meniscus, proteoglycan content is low during the development of collagen fibers and progressively increases with aging. In vitro, fibrochondrocytes produce glycosaminoglycans (GAGs) early in culture, in contrast to native tissue, where they are deposited after collagen fibers have formed. This difference in the timing of GAG production hinders the formation of a mature fiber network in such in vitro models. In this study, we removed GAGs from collagen gel-based tissue engineered constructs using chondroitinase ABC (cABC) and evaluated the effect on the formation and alignment of collagen fibers and the subsequent effect on tensile and compressive mechanical properties. Removal of GAGs during maturation of in vitro constructs improved collagen fiber alignment in tissue engineered meniscus constructs. Additionally, removal of GAGs during maturation improved fiber alignment without compromising compressive strength, and this removal improved not only fiber alignment and formation but also tensile properties. The increased fiber organization in cABC-treated groups also appeared to influence the size, shape, and location of defects in these constructs, suggesting that treatment may prevent the propagation of large defects under loading. This data gives another method of modulating the ECM for improved collagen fiber formation and mechanical properties in tissue engineered constructs.

A Knitted PET Patch Enhances the Maturation of Regenerated Tendons in Bridging Reconstruction of Massive Rotator Cuff Tears in a Rabbit Model

Background: Although nondegradable synthetic grafts for bridging reconstruction of massive rotator cuff tears (MRCTs) have shown satisfactory clinical outcomes, their function and details on graft-tendon healing and enthesis regeneration have not been fully studied. Hypothesis: The knitted polyethylene terephthalate (PET) patch as a nondegradable synthetic graft could provide sustained mechanical support, facilitating enthesis and tendon regeneration in the treatment of MRCTs. Study Design: Controlled laboratory study. Methods: A knitted PET patch was fabricated for bridging reconstruction (PET group) in a New Zealand White rabbit model of MRCTs (negative control group), and an autologous Achilles tendon was used as a control (autograft group). The animals were sacrificed, and tissue samples were harvested for gross observation as well as histological and biomechanical analyses at 4, 8, and 12 weeks postoperatively. Results: Histological analysis showed no significant difference in the graft-bone interface score between the PET and autograft groups at 4, 8, and 12 weeks postoperatively. Interestingly, in the PET group, Sharpey-like fibers were observed at 8 weeks, while fibrocartilage formation and the ingrowth of chondrocytes were recognized at 12 weeks. Meanwhile, the tendon maturing score was significantly higher in the PET group than in the autograft group (19.7 ± 1.5 vs 15.3 ± 1.2, respectively; P = .008) at 12 weeks, with parallel-oriented collagen fibers around the knitted PET patch. Moreover, the ultimate failure load of the PET group was similar to that of a healthy rabbit tendon at 8 weeks (125.6 ± 13.6 vs 130.8 ± 28.6 N, respectively; P > .05) and no different from that of the autograft group at 4, 8, and 12 weeks. Conclusion: The knitted PET patch could not only immediately reconstruct the mechanical support for the torn tendon postoperatively in the rabbit model of MRCTs but also enhanced maturation of the regenerated tendon by fibrocartilage formation and improved the organization of collagen fibers. Herein, the knitted PET patch could be a promising candidate graft adopted in bridging reconstruction of MRCTs. Clinical Relevance: A nondegradable knitted PET patch can safely bridge MRCTs with satisfactory mechanical strength and the promotion of tissue regeneration.

Organ- and function-specific anatomical organization of vagal fibers supports fascicular vagus nerve stimulation

Vagal fibers travel inside fascicles and form branches to innervate organs and regulate organ functions. Existing vagus nerve stimulation (VNS) therapies activate vagal fibers non-selectively, often resulting in reduced efficacy and side effects from non-targeted organs. The transverse and longitudinal arrangement of fibers inside the vagal trunk with respect to the functions they mediate and organs they innervate is unknown, however it is crucial for selective VNS. Using micro-computed tomography imaging, we tracked fascicular trajectories and found that, in swine, sensory and motor fascicles are spatially separated cephalad, close to the nodose ganglion, and merge caudad, towards the lower cervical and upper thoracic region; larynx-, heart- and lung-specific fascicles are separated caudad and progressively merge cephalad. Using quantified immunohistochemistry at single fiber level, we identified and characterized all vagal fibers and found that fibers of different morphological types are differentially distributed in fascicles: myelinated afferents and efferents occupy separate fascicles, myelinated and unmyelinated efferents also occupy separate fascicles, and small unmyelinated afferents are widely distributed within most fascicles. We developed a multi-contact cuff electrode to accommodate the fascicular structure of the vagal trunk and used it to deliver fascicle-selective cervical VNS in anesthetized and awake swine. Compound action potentials from distinct fiber types, and physiological responses from different organs, including laryngeal muscle, cough, breathing, and heart rate responses are elicited in a radially asymmetric manner, with consistent angular separations that agree with the documented fascicular organization. These results indicate that fibers in the trunk of the vagus nerve are anatomically organized according to functions they mediate and organs they innervate and can be asymmetrically activated by fascicular cervical VNS.

Revealing and manipulating the structure of mitotic chromosomes using atomic force microscopy

Although the mitotic cell cycle has been extensively characterized, very little is known about the highly condensed structure of metaphase chromosomes. The hierarchical organization of the chromatin fibers and their extrusion into complex loop structures by the Structural Maintenance of Chromosome (SMC) proteins are widely acknowledged features of the chromosomal architecture; however, the small size and complex topology of these structures are hindering their unequivocal detection and characterization. Even though Atomic Force Microscopy (AFM) has the potential to access these features, chromosome deformability and susceptibility to alterations to their external environment have so far limited the application of this technique. In the present work we overcome these issues and employ AFM to study the structure of human metaphase chromosomes in a liquid environment that closely resembles their physiological conditions, without the use of fixation agents and enzymatic treatments. Our analysis reveals the finer details of the unperturbed chromosome structure, shedding light on the organization of the chromatin fiber network and testing its stability against changes in the environment ionic conditions. Moreover, by complementing the image analysis with AFM-based Force Spectroscopy we probe the response of mitotic chromosomes to nanoscale deformations in the presence and absence of specific SMC proteins, namely Condensin I and II, thereby providing new insights into their contribution to the chromosome structural stability. The presented results enrich our current understanding of chromosome organization and provide new valuable tools to study the structure of mitotic chromosomes in both healthy and pathological conditions.

The second extracellular domain of connexin 50 is important for in cell adhesion, lens differentiation, and adhesion molecule expression

Connexin (Cx)-forming channels play essential roles in maintaining lens homeostasis and transparency. We showed here channel-independent roles of Cx50 in cell-cell adhesion and confirmed the second extracellular (E2) domain as a critical domain for cell adhesion function. We found that cell adhesion decreased in cells expressing chimeric Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with the Cx50 (E2) domain. This function is Cx channel-independent and Cx50 E2 domain-dependent cell adhesion acting in both homotypic and heterotypic manners. In addition, we generated eight site mutations of unique residues between Cx50 and the other two lens Cxs and found that mutation of any one of the residues abolished the adhesive function. Moreover, expression of adhesive-impaired mutants decreased adhesion-related proteins, N-cadherin and β-catenin. Expression of the adhesion-impaired Cx50W188P mutant in embryonic chick lens caused enlarged extracellular spaces, distorted fiber organization, delayed nuclear condensation, and cortical cataracts. In summary, the results from both invitro and invivo studies demonstrate the importance of the adhesive function of Cx50 in the lens.

Utilizing micro-CT imaging to define collagen modeling in murine aorta.

We aim to demonstrate local aortic tissue biodynamics via collagen and elastin fiber remodeling in murine aorta at varying distension using microcomputed tomography (microCT). Ten week-old C57BL/6 mice were injected via tail vein with in-vivo collagen hybridizing peptide, which reforms the triple-helix structure of denatured collagen, and is visualized via fluorescein tag. Careful dissection and removal of the thoracoabdominal aorta was performed, which was pressurized with formalin at varying distensions. Samples were then prepared via heavy-metal staining, and fixed in epoxy resin. These were subsequently imaged at the microCT scanner at University of Chicago, as well as at the 2-BM-B beamline using the Advanced Photon Source at Argonne National Laboratory to obtain micron and sub-micron resolution images and thus provide multi-level views of the tissue. Clear visualization of distinct collagen and elastin layers was observed with microCT. The images also clearly illustrate the effect on individual collagen and elastin fiber organization created by overdistension and shear-stress on the aorta. Clear demonstration of the geometric structure of aortic elastin fibers was seen upon initial segmentation. Preliminary experiments demonstrate that microCT provides an excellent visualization of murine aorta at micron and submicron levels. Next steps will be to segment and mesh this data in order to simulate local tissue responses to stress via finite element analysis. This method will produce a multi-scale imaging model of aortic failure.

Confocal reflectance microscopy for mapping collagen fiber organization in the vitreous gel of the eye.

Vitreous collagen structure plays an important role in ocular mechanics. However, capturing this structure with existing vitreous imaging methods is hindered by the loss of sample position and orientation, low resolution, or a small field of view. The objective of this study was to evaluate confocal reflectance microscopy as a solution to these limitations. Intrinsic reflectance avoids staining, and optical sectioning eliminates the requirement for thin sectioning, minimizing processing for optimal preservation of the natural structure. We developed a sample preparation and imaging strategy using ex vivo grossly sectioned porcine eyes. Imaging revealed a network of uniform diameter crossing fibers (1.1 ± 0.3 µm for a typical image) with generally poor alignment (alignment coefficient = 0.40 ± 0.21 for a typical image). To test the utility of our approach for detecting differences in fiber spatial distribution, we imaged eyes every 1 mm along an anterior-posterior axis originating at the limbus and quantified the number of fibers in each image. Fiber density was higher anteriorly near the vitreous base, regardless of the imaging plane. These data demonstrate that confocal reflectance microscopy addresses the previously unmet need for a robust, micron-scale technique to map features of collagen networks in situ across the vitreous.

Three-dimensional remodeling of collagen fibers within cervical tissues in pregnancy

The cervix is a collagen-rich connective tissue that must remain closed during pregnancy while undergoing progressive remodeling in preparation for delivery, which begins before the onset of the preterm labor process. Therefore, it is important to resolve the changes of collagen fibers during cervical remodeling for the prevention of preterm labor. Herein, we assessed the spatial organization of collagen fibers in a three-dimensional (3D) context within cervical tissues of mice on day 3, 9, 12, 15 and 18 of gestation. We found that the 3D directional variance, a novel metric of alignment, was higher on day 9 than that on day 3 and then gradually decreased from day 9 to day 18. Compared with two-dimensional (2D) approach, a higher sensitivity was achieved from 3D analysis, highlighting the importance of truly 3D quantification. Moreover, the depth-dependent variation of 3D directional variance was investigated. By combining multiple 3D directional variance-derived metrics, a high level of classification accuracy was acquired in distinguishing different periods of pregnancy. These results demonstrate that 3D directional variance is sensitive to remodeling of collagen fibers within cervical tissues, shedding new light on highly-sensitive, early detection of preterm birth (PTB).

Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against αv and β3 but not α5 or β1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human αvβ3 and αvβ6 integrins, but not α5β1 integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an αv-selective integrin agonist. This study provides evidence that cell surface αv-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

Severe Burn Injury Significantly Alters the Gene Expression and m6A Methylation Tagging of mRNAs and lncRNAs in Human Skin.

N6-methyladenosine (m6A) modulates RNA metabolism and functions in cell differentiation, tissue development, and immune response. After acute burns, skin wounds are highly susceptible to infection and poor healing. However, our understanding of the effect of burn injuries on m6A methylation and their potential mechanism is still limited. Human m6A-mRNA&lncRNA Epitranscriptomic microarray was used to obtain comprehensive mRNA and lncRNA transcriptome m6A profiling and gene expression patterns after burn injuries in human skin tissue. Bioinformatic and functional analyses were conducted to find molecular functions. Microarray profiling showed that 65 mRNAs and 39 lncRNAs were significantly hypermethylated; 5492 mRNAs and 754 lncRNAs were significantly hypomethylated. Notably, 3989 hypomethylated mRNAs were down-expressed and inhibited many wound healing biological processes and pathways including in the protein catabolic process and supramolecular fiber organization pathway; 39 hypermethylated mRNAs were up-expressed and influenced the cell surface receptor signaling pathway and inflammatory response. Moreover, we validated that m6A regulators (METTL14, METTL16, ALKBH5, FMR1, and HNRNPC) were significantly downregulated after burn injury which may be responsible for the alteration of m6A modification and gene expression. In summary, we found that homeostasis in the skin was disrupted and m6A modification may be a potential mechanism affecting trauma infection and wound healing.

Nanosecond pulsed electric field suppresses growth and reduces multi-drug resistance effect in pancreatic cancer

Nanosecond pulsed electric fields (nsPEF) have been shown to exert anticancer effects; however, little is known about the mechanisms triggered in cancer cells by nanosecond-length pulses, especially when low, sub-permeabilization voltage is used. In this study, three human pancreatic cancer cell lines were treated with nsPEF and molecular changes at the cellular level were analyzed. Further, we assessed the efficacy of paclitaxel chemotherapy following nsPEF treatment and correlated that with the changes in the expression of multi-drug resistance (MDR) proteins. Finally, we examined the influence of nsPEF on the adhesive properties of cancer cells as well as the formation and growth of pancreatic cancer spheroids. Cell line response differed with the application of a 200 ns, 100 pulses, 8 kV/cm, 10 kHz PEF treatment. PEF treatment led to (1) the release of microvesicles (MV) in EPP85-181RDB cells, (2) electropermeabilization in EPP85-181RNOV cells and (3) cell shrinkage in EPP85-181P cells. The release of MV’s in EPP85-181RDB cells reduced the membrane content of P-gp and LRP, leading to a transient increase in vulnerability of the cells towards paclitaxel. In all cell lines we observed an initial reduction in size of the cancer spheroids after the nsPEF treatment. Cell line EPP85-181RNOV exhibited a permanent reduction in the spheroid size after nsPEF. We propose a mechanism in which the surface tension of the membrane, regulated by the organization of actin fibers, modulates the response of cancer cells towards nsPEF. When a membrane’s surface tension remains low, we observed some cells form protrusions and release MVs containing MDR proteins. In contrast, when cell surface tension remains high, the cell membrane is being electroporated. The latter effect may be responsible for the reduced tumor growth following nsPEF treatment.

Possibilities of multiphoton microscopy for the diagnosis of the vulvar lichen sclerosus

Vulvar lichen sclerosus (VLS) is a chronic and recurrent dermatosis of an inflammatory nature with severe focal atrophy of the skin. Connective tissue changes are polymorphic and are still not taken into account in histological diagnostics due to the difficulty of interpreting routine histological methods. In this work, we use multiphoton microscopy (MPM) as a new imaging technique that provides detailed information about the organization of collagen fibers in the dermis based on a non-linear second harmonic generation (SHG) process. To determine the degree of connective tissue damage in lichen sclerosus using standard histological techniques and to reveal the diagnostic capabilities of multiphoton microscopy. We studied 42 biopsies with a histopathological diagnosis of VLS and 10 biopsies of normal vulvar skin. Histological, histochemical and immunohistochemical evaluation was used in comparison with MPM data. Quantitative analysis included the determination of the thickness, length of collagen fibers and the average intensity of the SHG signal. A comprehensive study of the skin showed 4 groups of changes that can be regarded as the degree of the dermis damage: initial, mild, moderate, severe. The affected area at the initial and mild degree has subtle changes, however, it is reliably identified by quantitative analysis of the SHG signal. So, the initial degree is characterized by thin (1.3-1.8 µm) long (56-69 µm) collagen fibers, with a moderate degree, the fibers are thickened (3.4-4.3 µm) and fragmented (22-37 µm). The affected area in moderate and severe cases undergoes homogenization, which is associated with the deposition of extremely thin (0.6-0.9 μm) short (16-28 μm) collagen fibers and the expression of type V collagen. Multiphoton microscopy in the second harmonic generation mode is a reliable method for identifying collagen fibers in tissues. The study made it possible to identify 4 degrees of the dermis damage in vulvar lichen sclerosus.

Гістологічна характеристика експериментальних ран м’яких тканин стегна щурів та роль IFN-γ в динаміці їх загоєння

The purpose of the study was to evaluate the histomorphological changes in samples of the wound canal of the soft tissues of the femur of rats and to study the role of interferon-gamma (IFN-γ) in the dynamics of wound healing. Materials and methods. The study was conducted on 24 Wistar rats. Animals were divided into two groups – intact (6 rats) and experimental (18 rats). Animals of the experimental group were used to simulate wounds. Rats were euthanized on the 10th, 20th, and 40th days (6 animals for each time) after wound simulation. Blood for the study was taken by the method of open cardiac puncture. The levels of INF-γ in the blood serum of animals were determined by enzyme immunoassay. Histological processing of the samples was carried out according to generally accepted methods, the sections were stained with hematoxylin and eosin, as well as picrofuchsin according to Van Gieson. Results and discussion. The work revealed changes in the expression of IFN-γ in the blood serum of animals with wounds: an increase in the levels of IFN-γ on the 10th and 20th days after modeling wounds compared to the levels of rats in the intact group (p&lt;0.05). A decrease in the levels of IFN-γ on the 40th day of the experiment compared to a similar level of this cytokine on the 10th and 20th days after wound simulation was shown (p&lt;0.05). The results of histological studies show signs of the proliferative phase in the form of a significant number of fibroblasts and newly formed vessels, as well as the beginning of the remodeling stage in the form of the organization of collagen fibers in parallel bundles in the wound canals in different areas within 10 days. On the 20th day, the absence of inflammatory cells in the preparations of the damaged areas is noted. Also at the tissue level at this time, signs of the remodeling phase were revealed: a significant decrease in the number of blood vessels and fibroblasts was observed, the connective tissue in the areas acquired a mature appearance in the form of dense layers with single fibrocytes. In the injury zone on the 40th day of the experiment, scars from mature connective tissue were noted. Conclusion. In an experimental study, we showed the morphological and physiological features of the healing of soft tissue injuries in normal rats. The established structural features of wound areas at different stages of healing and the dynamics of IFN-γ release allow us to determine its important role not only in inflammation, but also in the stages of proliferation and remodeling. Determination of the concentration of IFN-γ may be an informative indicator at all stages of repair in the process of healing soft tissue injuries in humans, subjected to further clinical studies

Selenium Deficiency Dysregulates One-Carbon Metabolism in Nutritional Muscular Dystrophy of Chicks

BackgroundNutritional muscular dystrophy (NMD) in animals is induced by dietary selenium (Se) deficiency. ObjectivesThis study was conducted to explore the underlying mechanism of Se deficiency–induced NMD in broilers. MethodsOne-day-old male Cobb broilers (n = 6 cages/diet, 6 birds/cage) were fed a Se-deficient diet (Se-Def, 47 μg Se/kg) or the Se-Def supplemented with 0.3 mg Se/kg (control) for 6 wk. Thigh muscles of broilers were collected at week 6 for measuring Se concentration, histopathology, and transcriptome and metabolome assays. The transcriptome and metabolome data were analyzed with bioinformatics tools and other data were analyzed with Student’s t tests. ResultsCompared with the control, Se-Def induced NMD in broilers, including reduced (P < 0.05) final body weight (30.7%) and thigh muscle size, reduced number and cross-sectional area of fibers, and loose organization of muscle fibers. Compared with the control, Se-Def decreased (P < 0.05) the Se concentration in the thigh muscle by 52.4%. It also downregulated (P < 0.05) GPX1, SELENOW, TXNRD1-3, DIO1, SELENOF, H, I, K, M, and U by 23.4–80.3% in the thigh muscle compared with the control. Multi-omics analyses indicated that the levels of 320 transcripts and 33 metabolites were significantly altered (P < 0.05) in response to dietary Se deficiency. Integrated transcriptomics and metabolomics analysis revealed that one-carbon metabolism, including the folate and methionine cycle, was primarily dysregulated by Se deficiency in the thigh muscles of broilers. ConclusionsDietary Se deficiency induced NMD in broiler chicks, potentially with the dysregulation of one-carbon metabolism. These findings may provide novel treatment strategies for muscle disease.

Comparison of biopolymer scaffolds for the fabrication of skin substitutes in a porcine wound model.

This study compared three acellular scaffolds as templates for the fabrication of skin substitutes. A collagen-glycosaminoglycan (C-GAG), a biodegradable polyurethane foam (PUR) and a hybrid combination (PUR/C-GAG) were investigated. Scaffolds were prepared for cell inoculation. Fibroblasts and keratinocytes were serially inoculated onto the scaffolds and co-cultured for 14 days before transplantation. Three pigs each received four full-thickness 8cm × 8cm surgical wounds, into which a biodegradable temporising matrix (BTM) was implanted. Surface seals were removed after integration (28 days), and three laboratory-generated skin analogues and a control split-thickness skin graft (STSG) were applied for 16 weeks. Punch biopsies confirmed engraftment and re-epithelialisation. Biophysical wound parameters were also measured and analysed. All wounds showed greater than 80% epithelialisation by day 14 post-transplantation. The control STSG displayed 44% contraction over the 16 weeks, and the test scaffolds, C-GAG 64%, Hybrid 66.7% and PUR 67.8%. Immunohistochemistry confirmed positive epidermal keratins and basement membrane components (Integrin alpha-6, collagens IV and VII). Collagen deposition and fibre organisation indicated the degree of fibrosis and scar produced for each graft. All scaffold substitutes re-epithelialised by 4 weeks. The percentage of original wound area for the Hybrid and PUR was significantly different than the STSG and C-GAG, indicating the importance of scaffold retainment within the first 3 months post-transplant. The PUR/C-GAG scaffolds reduced the polymer pore size, assisting cell retention and reducing the contraction of in vitro collagen. Further investigation is required to ensure reproducibility and scale-up feasibility.