Number Of Branch Points Research Articles

Segmentation and Evaluation of Corneal Nerves and Dendritic Cells From In Vivo Confocal Microscopy Images Using Deep Learning.

PurposeSegmentation and evaluation of in vivo confocal microscopy (IVCM) images requires manual intervention, which is time consuming, laborious, and non-reproducible. The aim of this research was to develop and validate deep learning–based methods that could automatically segment and evaluate corneal nerve fibers (CNFs) and dendritic cells (DCs) in IVCM images, thereby reducing processing time to analyze larger volumes of clinical images.MethodsCNF and DC segmentation models were developed based on U-Net and Mask R-CNN architectures, respectively; 10-fold cross-validation was used to evaluate both models. The CNF model was trained and tested using 1097 and 122 images, and the DC model was trained and tested using 679 and 75 images, respectively, at each fold. The CNF morphology, number of nerves, number of branching points, nerve length, and tortuosity were analyzed; for DCs, number, size, and immature–mature cells were analyzed. Python-based software was written for model training, testing, and automatic morphometric parameters evaluation.ResultsThe CNF model achieved on average 86.1% sensitivity and 90.1% specificity, and the DC model achieved on average 89.37% precision, 94.43% recall, and 91.83% F1 score. The interclass correlation coefficient (ICC) between manual annotation and automatic segmentation were 0.85, 0.87, 0.95, and 0.88 for CNF number, length, branching points, and tortuosity, respectively, and the ICC for DC number and size were 0.95 and 0.92, respectively.ConclusionsOur proposed methods demonstrated reliable consistency between manual annotation and automatic segmentation of CNF and DC with rapid speed. The results showed that these approaches have the potential to be implemented into clinical practice in IVCM images.Translational RelevanceThe deep learning–based automatic segmentation and quantification algorithm significantly increases the efficiency of evaluating IVCM images, thereby supporting and potentially improving the diagnosis and treatment of ocular surface disease associated with corneal nerves and dendritic cells.

Conductive Collagen-Based Hydrogel Combined With Electrical Stimulation to Promote Neural Stem Cell Proliferation and Differentiation.

Injectable biomimetic hydrogels are a promising strategy for enhancing tissue repair after spinal cord injury (SCI) by restoring electrical signals and increasing stem cell differentiation. However, fabricating hydrogels that simultaneously exhibit high electrical conductivities, excellent mechanical properties, and biocompatibility remains a great challenge. In the present study, a collagen-based self-assembling cross-linking polymer network (SCPN) hydrogel containing poly-pyrrole (PPy), which imparted electroconductive properties, is developed for potential application in SCI repair. The prepared collagen/polypyrrole (Col/PPy)-based hydrogel exhibited a continuous and porous structure with pore sizes ranging from 50 to 200 μm. Mechanical test results indicated that the Young’s moduli of the prepared hydrogels were remarkably enhanced with PPy content in the range 0–40 mM. The conductivity of Col/PPy40 hydrogel was 0.176 ± 0.07 S/cm, which was beneficial for mediating electrical signals between tissues and accelerating the rate of nerve repair. The investigations of swelling and degradation of the hydrogels indicated that PPy chains interpenetrated and entangled with the collagen, thereby tightening the network structure of the hydrogel and improving its stability. The cell count kit-8 (CCK-8) assay and live/dead staining assay demonstrated that Col/PPy40 coupled with electrical simulation promoted the proliferation and survival of neural stem cells (NSCs). Compared with the other groups, the immunocytochemical analysis, qPCR, and Western blot studies suggested that Col/PPy40 coupled with ES maximally induced the differentiation of NSCs into neurons and inhibited the differentiation of NSCs into astrocytes. The results also indicated that the neurons in ES-treated Col/PPy40 hydrogel have longer neurites (170.8 ± 37.2 μm) and greater numbers of branch points (4.7 ± 1.2). Therefore, the prepared hydrogel system coupled with ES has potential prospects in the field of SCI treatment.

Hyaluronic Acid with Bone Substitutes Enhance Angiogenesis In Vivo.

Introduction: The effective induction of angiogenesis is directly related to the success of bone-substitute materials (BSM) for maxillofacial osseous regeneration. Therefore, the addition of pro-angiogenic properties to a commercially available bovine bone-substitute material in combination with hyaluronic acid (BSM+) was compared to the same bone-substitute material without hyaluronic acid (BSM) in an in-vivo model. Materials and Methods: BSM+ and BSM were incubated for six days on the chorioallantoic membrane (CAM) of fertilized chicken eggs. Microscopically, the number of vessels and branching points, the vessel area and vessel length were evaluated. Subsequently, the total vessel area and brightness integration were assessed after immunohistochemical staining (H&E, alphaSMA). Results: In the BSM+ group, a significantly higher number of vessels (p < 0.001), branching points (p = 0.001), total vessel area (p < 0.001) as well as vessel length (p = 0.001) were found in comparison to the BSM group without hyaluronic acid. Immunohistochemically, a significantly increased total vessel area (p < 0.001 for H&E, p = 0.037 for alphaSMA) and brightness integration (p = 0.047) for BSM+ in comparison to the native material were seen. Conclusions: The combination of a xenogenic bone-substitute material with hyaluronic acid significantly induced angiogenesis in vivo. This might lead to a faster integration and an improved healing in clinical situations.

Unbiased analysis of mouse brain endothelial networks from two- or three-dimensional fluorescence images.

.Significance: A growing body of research supports the significant role of cerebrovascular abnormalities in neurological disorders. As these insights develop, standardized tools for unbiased and high-throughput quantification of cerebrovascular structure are needed.Aim: We provide a detailed protocol for performing immunofluorescent labeling of mouse brain vessels, using thin () or thick (50 to ) tissue sections, followed respectively by two- or three-dimensional (2D or 3D) unbiased quantification of vessel density, branching, and tortuosity using digital image processing algorithms.Approach: Mouse brain sections were immunofluorescently labeled using a highly selective antibody raised against mouse Cluster of Differentiation-31 (CD31), and 2D or 3D microscopy images of the mouse brain vasculature were obtained using optical sectioning. An open-source toolbox, called Pyvane, was developed for analyzing the imaged vascular networks. The toolbox can be used to identify the vasculature, generate the medial axes of blood vessels, represent the vascular network as a graph, and calculate relevant measurements regarding vascular morphology.Results: Using Pyvane, vascular parameters such as endothelial network density, number of branching points, and tortuosity are quantified from 2D and 3D immunofluorescence micrographs.Conclusions: The steps described in this protocol are simple to follow and allow for reproducible and unbiased analysis of mouse brain vascular structure. Such a procedure can be applied to the broader field of vascular biology.

MO675: A New in Vivo Multi-Photon Microscopy Based Approach to Study the Peritoneal Membrane Changes Induced by Peritoneal Dialysis

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is a renal replacement therapy that allows the elimination of metabolic waste products and excess body fluid through the peritoneal membrane. The exposure to PD solution contributes to membrane aging and fibrosis resulting in ultrafiltration and clearance failure. The glucose, used as an osmotic factor, in PD dialysate triggers several processes involved in the pathogenesis of peritoneal fibrosis, angiogenesis and epithelial to mesenchymal transition. Studies on natural extracts, such as Oleuropein (Ole), a powerful antioxidant with remarkable antifibrotic and protective effects on the peritoneal membrane, are currently being validated. This study aims to develop a method based on multi-photon microscopy to study the physiology of the peritoneal membrane during dialysis exchange and to validate in vivo the effects of the Ole in animal models of fibrosis undergoing dialysis treatment. METHOD Multi-photon microscopy allows in vivo evaluation of the microcirculation that supply the peritoneum and also to study the framework of mesothelial cells and their underlying layer of collagen fibers that contributes to the sub-mesothelial space. We have implemented the surgical procedure in order to optimize the stability of a flap of parietal peritoneum to directly observe at the scope. With this approach the peritoneal membrane is evaluable at baseline condition and during exposure to dialysate solutions. RESULTS Our method allows building a three-dimensional render of the peritoneal membrane, with the evaluation of all the single layers without the use of specific markers. In this way we could assess specifically the phenomena induced by the fibrotic process: the thickening of the sub-mesothelial interstitium and the greater density of the vascular network. Furthermore, in vivo measurements of flow in the vessels of microcirculation (arterioles, capillaries, post-capillary venules) determine that exposure to hypotonic solutions increases significantly the flow in large-diameter vessels due to better permeability and hemodilution. Finally in order to evaluate the possibility to detect morphological and functional changes in pathogenic model we have used the well-established model of peritoneal fibrosis derived by 15 days’ long exposure of the peritoneal membrane with 3.86% glucose dialysate. Whth this approach we could detect the significant increase in the parameters of cellularity, vascularization, fibrosis and thickening of collagen fibers. In order to test the sensibility of our approach to the evaluation of the peritoneal membrane senescence parameters, we tested the effect of Ole in preventing the damage induced by high glucose–containing dialysate. Ole reduced both the thickness and the organization of the collagen fibers and the vascular network, including the number of branch points. CONCLUSION The developed method has potential for a dynamic and reliable in vivo approach. Studies are ongoing to validate the effects of drugs and dialysates with different osmotic and electrolytic compositions on the dialysis capacities of the peritoneal membrane and on the blood flow in peritoneal capillaries. This method offers great potential for testing new pharmacological approaches aimed at preserving the structural and functional integrity of the peritoneum and for the validation of substances, such as natural extracts with beneficial effects against the damage induced in the long term by conventional dialysis solutions.

Analysis of Cerebrovascular Architecture in Experimental Models of Hypertension and Chronic Neuroendocrine Stress

Cerebral small vessel disease and Alzheimer’s disease are leading causes of dementia. Psychological/social stress and associated hypertension are important drivers of brain vascular dysfunction and impaired perfusion. We set out to investigate if chronic neuroendocrine stress and hypertension have adverse effects on cerebrovascular architecture of various brain regions in two rodent models: 1) a rat model where HPA axis activation and hypertension were induced by brain derived neurotrophic factor (BDNF) overexpression in the paraventricular nucleus of the hypothalamus (PVN) for 5 wks, and 2) in a mouse model, where blood pressure and/or corticosterone levels were elevated by subcutaneous infusions of angiotensin II (AngII, 1.5mg/kg/day), adrenocorticotropic hormone (ACTH, 30ug/kg/day) or a combination of AngII and ACTH for 4 weeks. Cerebrovasculature was labeled by transcardiac perfusion of a liposome‑encapsulated fluorescent DiI dye. Brains were fixed in 4% PFA and transparency was increased with the SeeDB method. Vasculature of the hippocampus (HPC), sensory cortex (CTX), and PVN was evaluated using 2‐photon microscopy in 2mm thick coronal brain slices. 3D model of the vasculature was created by Imaris software (Oxford Instruments) yielding parameters such as vascular segment numbers, lengths, diameters, and number of branch points. 3D Euclidean distance mapping (EDM) was used to determine distribution of distances to nearest vessels (DNVs) within the extravascular space as a measure of accessibility to blood supply. We found that vascular densities were markedly elevated in the PVN relative to other brain regions in both rats and mice. In mice, HPC vascular density was significantly lower relative to CTX, whereas in rats, CTX and HPC densities were similar [vascular densities % (rats, mice): PVN:14.6±1.4, 17.0±0.6; CTX: 9.6±0.5, 8.6±0.4; HPC CA: 8.8±0.7, 6.2±0.3; HPC DG: 9.4±0.6, 5.4±0.3]. EDM analysis revealed that the proportion of short (&lt;15μm) vs long DNVs was the highest in the PVN in both rats and mice. Distribution of DNVs shifted to longer distances in the CTX, and the proportion of short vs long DNVs was the lowest in HPC regions indicating an increased vulnerability to hypoperfusion. Interestingly, neuroendocrine stress and hypertension failed to significantly affect vascular densities and DNV statistics in either rats or mice in the analyzed brain regions. However, there was a notable sex difference in distribution of DNVs in HPC CA regions with male rats having a higher proportion of longer distances than females. In addition, analysis of vascular diameter distribution revealed that BDNF treatment decreased vessel diameters in the PVN of both male and female rats, and in the CTX of male, but not female rats. In summary, we found significant region‐specific variability in vascular structure, with the PVN demonstrating a uniquely dense vascular network. Cerebrovascular architecture was fairly resilient to treatments resembling chronic stress‐induced neuroendocrine and cardiovascular responses, but sex differences in HPC vasculature may promote a higher sensitivity to hypoperfusion in male vs female rats.

Removal of Proteins and Its Effect on Molecular Structure and Properties of Natural Rubber

Removal of protein from natural rubber was carried out via deproteinization of natural rubber in the latex stage. Then, its effect on the molecular structure and properties of natural rubber was investigated. Urea, SDS, and acetone were used as denaturing agent, a surfactant, and a polar solvent in the deproteinization, respectively. Various deproteinized natural rubbers were obtained after one, two, and three times of centrifugation with and without acetone, namely DPNR1, DPNR2, DPNR3, DPNR1-A, DPNR2-A, and DPNR3-A. It was found that the total protein content significantly decreased as increasing the number of centrifugations; however, the total fatty acid contents showed a slight decrease. Structural characteristics analyzed by nuclear magnetic resonance spectroscopy indicated no changes in the chemical structure of natural rubber after deproteinization. However, the removal of proteins significantly enhanced the resolution of the NMR signals. Gel content and tensile properties of natural rubber showed a decrease in the removal of proteins, which was associated with the decrease in the number of the inherent branching points formed in natural rubber.

Common Microscale and Macroscale Principles of Connectivity in the Human Brain.

The brain requires efficient information transfer between neurons and large-scale brain regions. Brain connectivity follows predictable organizational principles. At the cellular level, larger supragranular pyramidal neurons have larger, more branched dendritic trees, more synapses, and perform more complex computations; at the macroscale, region-to-region connections display a diverse architecture with highly connected hub areas facilitating complex information integration and computation. Here, we explore the hypothesis that the branching structure of large-scale region-to-region connectivity follows similar organizational principles as the neuronal scale. We examine microscale connectivity of basal dendritic trees of supragranular pyramidal neurons (300+) across 10 cortical areas in five human donor brains (1 male, 4 female). Dendritic complexity was quantified as the number of branch points, tree length, spine count, spine density, and overall branching complexity. High-resolution diffusion-weighted MRI was used to construct white matter trees of corticocortical wiring. Examining complexity of the resulting white matter trees using the same measures as for dendritic trees shows heteromodal association areas to have larger, more complex white matter trees than primary areas (p < 0.0001) and macroscale complexity to run in parallel with microscale measures, in terms of number of inputs (r = 0.677, p = 0.032), branch points (r = 0.797, p = 0.006), tree length (r = 0.664, p = 0.036), and branching complexity (r = 0.724, p = 0.018). Our findings support the integrative theory that brain connectivity follows similar principles of connectivity at neuronal and macroscale levels and provide a framework to study connectivity changes in brain conditions at multiple levels of organization.SIGNIFICANCE STATEMENT Within the human brain, cortical areas are involved in a wide range of processes, requiring different levels of information integration and local computation. At the cellular level, these regional differences reflect a predictable organizational principle with larger, more complexly branched supragranular pyramidal neurons in higher order regions. We hypothesized that the 3D branching structure of macroscale corticocortical connections follows the same organizational principles as the cellular scale. Comparing branching complexity of dendritic trees of supragranular pyramidal neurons and of MRI-based regional white matter trees of macroscale connectivity, we show that macroscale branching complexity is larger in higher order areas and that microscale and macroscale complexity go hand in hand. Our findings contribute to a multiscale integrative theory of brain connectivity.

Wave-optics investigation of branch-point density

We use wave-optics simulations to investigate branch-point density (i.e., the number of branch points within the pupil-phase function) in terms of the grid sampling. The goal for these wave-optics simulations is to model plane-wave propagation through homogeneous turbulence, both with and without the effects of a finite inner scale modeled using a Hill spectrum. In practice, the grid sampling provides a gauge for the amount of branch-point resolution within the wave-optics simulations, whereas the Rytov number, Fried coherence diameter, and isoplanatic angle provide parameters to setup and explore the associated deep-turbulence conditions. Via Monte Carlo averaging, the results show that without the effects of a finite inner scale, the branch-point density grows without bound with adequate grid sampling. However, the results also show that as the inner-scale size increases, this unbounded growth (1) significantly decreases as the Rytov number, Fried coherence diameter, and isoplanatic angle increase in strength and (2) saturates with adequate grid sampling. These findings imply that future developments need to include the effects of a finite inner scale to accurately model the multifaceted nature of the branch-point problem in adaptive optics.

Step‐Growth Polymerized Systems of General Type “AfiBgi”: A Method to Calculate the Bivariate (Molecular Size) × (Path Length) Distribution

AbstractStep‐growth polymerized systems of general type “AfiBgi” are considered. The monomers bear one or more reactive groups of type A and/or B. An A group on one monomeric unit might react with a B group on another unit, thus coupling the two units. This process repeats itself randomly, and at a given degree of conversion, a wide range of polymeric molecules has formed, differing in both molecular size and in branching structure, determined by the laws of probability. In a slice of the molecular size distribution, all polymeric molecules have the same size, but differ in number and position of branching points. A method is presented to calculate the path length distribution for each such slice. Here, path length is the number of chemical bonds in the path connecting two monomeric units in the molecule. From the path length distribution, the light scattering function is calculated.

COMBINING IN VITRO AND IN OVO ASSAYS TO SCREEN FOR ANTI-CANCER AND ANTI-ANGIOGENIC EFFECTS OF THE LEAF EXTRACTS OF Mallotus cumingii Mull.Arg. (EUPHORBIACEAE)

Treatment for cancer is often challenging and various interventions may have detrimental effects. Due to this, the development of less harmful alternatives such as herbal medicine is essential. The present study aims to determine the leaf phytoconstituents present and the bioactivities of Mallotus cumingii Müll.Arg against cancer cells through the utilization of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay and anti-angiogenesis through CAM or chorioallantoic membrane assay. The leaf extracts obtained three fractions namely, methanolic crude (MCME) extracts, hexane extracts (MCHE), and ethyl acetate extracts (MCEA), and were tested on HCT-116 (human colorectal cancer cell line) for in vitro cytotoxicity, and blood vessel density and branching through in ovo CAM assay. Phytochemical analysis showed that the M. cumingii fractions contain phenolic compounds, terpenoids, cardiac glycosides, flavonoids, and saponins. For in vitro set-up, MCME of M. cumingii were separated into MCHE and MCEA partitions and were tested against HCT-116 and obtained an IC50 value of &lt; 30 μg/mL, which is deemed active in cytotoxicity. For in ovo set-up, two concentrations of each extract were applied to the duck eggs. Blood vessel density and number of branching points were measured through the ImageJ analysis. All extracts exhibited anti-angiogenic activity, either by decreasing blood vessel density or the number of branching points. Overall, the study demonstrates the potential of M. cumingii as a source of therapeutic agents.

Blue Light-Induced Retinal Neuronal Injury and Amelioration by Commercially Available Blue Light-Blocking Lenses.

Blue light exposure-induced retinal damage has been extensively studied. Although many in vitro studies have shown the benefits of blue light-blocking lenses (BBL) there have been few comprehensive in vivo studies to assess the effects of BBL. We investigated the influence of blue light exposure using light-emitting diodes on retinal histology and visual cortex neurons in rodents. We also considered whether retinal and cortical changes induced by blue light could be ameliorated with blue light-blocking lenses. A total of n = 24 (n = 6 in each group; control, light exposure without lenses, two different BBLs)) male Wistar rats were subjected to blue light exposure (LEDs, 450–500 lux) without or with BBLs (400–490 nm) for 28 days on a 12:12 h light–dark cycle. Histological analysis of retinae revealed apoptosis and necrosis of the retinal pigment epithelium (RPE), photoreceptors, and inner retina in the light exposure (LE) group, along with increase caspase-3 immunostaining in the ganglion cell layer (p < 0.001). BBL groups showed less caspase-3 immunostaining compared with the LE group (p < 0.001). V1-L5PNs (primary visual cortex layer 5 pyramidal neurons) demonstrated reduced branching and intersections points for apical (p < 0.001) and basal (p < 0.05) dendrites following blue light exposure. Blue light-blocking lenses significantly improved the number of basal branching points compared with the LE group. Our study shows that prolonged exposure to high levels of blue light pose a significant hazard to the visual system resulting in damage to the retina with the associated remodeling of visual cortex neurons. BBL may offer moderate protection against exposure to high levels of blue light.

DOP13 The Arborisation index: An MRI-based measure of mesenteric hyperaemia in Crohn’s Disease

Abstract Background Robust and sensitive therapeutic targets are key in effective management of Inflammatory Bowel Disease1. Mesenteric hyperaemia is a recognized sign of active disease and in cross-sectional image is described as the comb sign. Although it is subjectively described, no automated quantitative MRI-based measures have been developed. We aim to develop an automated methodology using contrast-less time of flight (TOF) Magnetic resonance angiography (MRA). Methods A MATLAB algorithm was developed to track the vessels on a 3D maximum intensity projection of a TOF MRA data set and calculate an arborization Index which is the number of branching points in the intrabdominal vessels (figure 1). 2D TOF scans were acquired in the transverse plane between the top of the hip joint and L4 vertebra using a 3T Ingenia Wide bore scanner (Philips, The Netherlands). The primary outcome was a comparison of the arborization index between Crohn’s disease (CD) and healthy volunteers (HV) groups. A planned sub-analysis was undertaken across CD and HV matched for BMI to investigate the effect of visceral fat on the arborization index. Repeated measures were undertaken to evaluate the variability of the quantification method. No contrast agents were used for the TOF MRA scans. Biological variations within each group and test-retest repeatability were assessed using the coefficient of variation (CV). Statistical analysis with unpaired, two-tailed t-tests were conducted and differences were considered significant when the p-value ≤0.05. All absolute values are presented as mean ±standard deviation (SD). Results In this prospective pilot study, 7 CD patients (C-Reactive Protein=5.2±6.1 mg/L, Faecal Calprotectin 611±981μg/g, BMI=23±3 kg/m2) and 15 HVs (BMI=29±7 kg/m2) were recruited. Patients showed a significantly higher arborization index when compared to HVs (mean arborization in HV=94±21 and CD=139±26; p-value=0.002). The difference in arborization index persisted in a sub-analysis of 7 HVs (BMI=24±2 kg/m2) and 7 CD patients (mean arborization in matched HVs=101±22 vs mean index in CD=139±26; p=0.01) (Figure 2). The CV was 23% for HVs and 18% for CD indicating biological variation. Test-retest variability calculated from multiple TOF scans of the same subjects gave a mean CV of 6±5% for both groups combined. Conclusion Our preliminary data suggest that the arborization index may be a useful measure of hypervascularity and hence intestinal inflammation in Inflammatory Bowel Disease. Further validation to standard disease activity measures is needed across larger cohorts to better investigate the utility of this potential biomarker as a non-invasive measure of disease activity and its reversibility to IBD therapies. Reference 1.Turner,D.,et al.Gastro.2021;160(5):1570–1583.

Angiogenesis Promotion by Combined Administration of DFO and Vein Endothelial Cells Using Injectable, Biodegradable, Nanocomposite Hydrogel Scaffolds.

Desferrioxamine (DFO) upregulates HIF-1α and stimulates expression of vascular endothelial growth factor (VEGF), thereby accelerating neovascularization. As DFO acts primarily upon surrounding vein endothelial cells to stimulate angiogenesis, the angiogenic efficacy of DFO could be reduced in severely injured tissues lacking a sufficient number of vein endothelial cells. We hypothesized that combined administration of DFO and vein endothelial cells is a promising tissue engineering approach for promoting neovascularization. In this study, we evaluated the applicability of this approach using injectable, biocompatible, biodegradable nanocomposite gels consisting of poly(dl-lactide-co-glycolide)-b-polyethylene glycol-b-poly(dl-lactide-co-glycolide) (PLGA-PEG-PLGA) copolymers and clay nanoparticle LAPONITE. The nanocomposites exhibited irreversible thermo-gelation in the presence of DFO, and the mechanical strength was strongly affected by the amount of DFO. The storage moduli of the gels increased with increasing amount of DFO. These results indicate that the interaction between DFO and LAPONITE works as physical cross-linking points and facilitates the formation of the gel network. The nanocomposite gels achieved sustained slow release of DFO due to interactions between DFO and LAPONITE. Human umbilical vein endothelial cells (HUVECs) cultured on DFO-loaded nanocomposite gels exhibited a higher degree of vascular tube formation than cells cultured on nanocomposite gels without DFO. Moreover, the number of branching points and the diameter of the blood vessels regenerated in the gels significantly increased with increasing DFO amount, indicating that DFO released from the gels facilitates vascular tube-forming capacity. As a proof of concept, we demonstrate that the combined administration of DFO and vein endothelial cells using nanocomposite gels promotes greater angiogenesis than DFO administration alone using the same gels by in vivo experiments, confirming the validity of our hypothesis. Considering the multiple advantages of nanocomposite gels with regard to potential vascularization capacity, certain biocompatibility, biodegradability, and injectable cell- and drug-delivery capacity, we concluded that the nanocomposite gels have potential utility as scaffolding biomaterials for vascularization in tissue engineering applications.

Fibrillar gel self-assembly via cononsolvency of amphiphilic polymer

HypothesisPolymer with amphiphilic repeating units bathed in the mixed binary solvent can exhibit fibrillar formation and gelation via cononsolvency effect. Mechanism of gelation will be highly dependent on the solvent’s interactions and morphology of the resulting fibrillar gel can be fine-tuned by changing binary solvent composition. ExperimentsAmphiphilic homopolymers dissolved in a mixture of two solvents with different affinities to monomeric units and to each other were modeled using dissipative particle dynamics. Morphological transitions in dilute and concentrated solutions were investigated depending on the solvent-cosolvent interaction and binary solvent composition. Fibrillar gel structure was characterized via calculation of fibril’s and pore’s diameters. FindingsAmphiphilic macromolecules can combine into fibrils and form a gel as a result of the cononsolvency caused by microsegregation with one or both components of a binary solvent. In the former, the fibrils are loose, in the latter - dense. For dilute solution, state diagram in terms of solvent/cosolvent miscibility and binary solvent composition is constructed and two regions with different fibrillar gels are distinguished. For concentrated solution, the conditions, governing fibrillar diameter, pore size, number and functionality of branching points, are highlighted. These findings would be useful for the controlling synthetic matrix morphologies in the tissue engineering.

Exposure to an enriched environment promotes dendritic remodelling in hippocampal neurons affected by endogenous depression

Neuronal plasticity is enhanced in an enriched environment (EE) with more sensory and social interaction. In an animal model of endogenous depression, we have previously shown that EE has positive effects on spatial memory and hippocampus synaptic plasticity. However, nothing is known about how EE influences dendritic remodelling in hippocampal neurons affected by endogenous depression. In depressed rats, the impact of EE on hippocampus neuronal morphology was examined. Neonatal clomipramine exposure from postnatal days (PND) 8-21 days induced endogenous depression. The depressed-like rats were exposed to an enriched environment for two weeks in adulthood. Brains were then collected, stained with a modified Golgi-cox technique and, the hippocampal CA1 dendritic arborisation was evaluated using the Neurolucida software. Depression resulted in the atrophy of CA1 hippocampal neurons. The number of branching points and the overall number of dendritic intersections were reduced in depressed rats,. Exposure to an enriched environment significantly increased dendritic branching and the total number of dendritic intersections in hippocampal CA1 pyramidal neurons. The hippocampal pyramidal neuronal morphology of depressed rats improved after exposure to environmental enrichment. Neuronal plasticity and the development of novel therapeutic strategy will be improved by a greater understanding of how the environment affects neuronal morphology in depressed states.

Myofibre injury induces capillary disruption and regeneration of disorganized microvascular networks.

Injury to skeletal muscle disrupts myofibres and their microvascular supply. While the regeneration of myofibres is well described, little is known of how the microcirculation is affected by skeletal muscle injury or its recovery during regeneration. Nevertheless, the microvasculature must also recover to restore skeletal muscle function. We aimed to define the nature of microvascular damage and time course of repair during muscle injury and regeneration induced by the myotoxin BaCl2 . To test the hypothesis that microvascular disruption occurred secondary to myofibre injury, isolated microvessels were exposed to BaCl2 or the myotoxin was injected into the gluteus maximus (GM) muscle of mice. In isolated microvessels, BaCl2 depolarized smooth muscle cells (SMCs) and endothelial cells while increasing intracellular calcium in SMCs but did not elicit death of either cell type. At 1day post-injury (dpi) of the GM, capillary fragmentation coincided with myofibre degeneration while arteriolar and venular networks remained intact; neutrophil depletion before injury did not prevent capillary damage. Perfused capillary networks reformed by 5dpi in association with more terminal arterioles and were dilated through 10dpi. With no change in microvascular area or branch point number in regenerating capillary networks, fewer capillaries aligned with myofibres and were no longer organized into microvascular units. By 21dpi, capillary orientation and microvascular unit organization were no longer different from uninjured GM. We conclude that following their disruption secondary to myofibre damage, capillaries regenerate as disorganized networks that remodel into microvascular units as regenerated myofibres mature. KEY POINTS: Skeletal muscle regenerates after injury; however, the nature of microvascular damage and repair is poorly understood. Here, the myotoxin BaCl2 , a standard experimental method of acute skeletal muscle injury, was used to investigate the response of the microcirculation to local injury of intact muscle. Intramuscular injection of BaCl2 induced capillary fragmentation with myofibre degeneration; arteriolar and venular networks remained intact. Direct exposure to BaCl2 did not kill microvascular endothelial cells or smooth muscle cells. Dilated capillary networks reformed by 5days post-injury (dpi) in association with more terminal arterioles. Capillary orientation remained disorganized through 10dpi. Capillaries realigned with myofibres and reorganized into microvascular units by 21dpi, which coincides with the recovery of vasomotor control and maturation of nascent myofibres. Skeletal muscle injury disrupts its capillary supply secondary to myofibre degeneration. Reorganization of regenerating microvascular networks accompanies the recovery of blood flow regulation.

Characterization of an Alzheimer disease-associated deletion in SORL1.

Greater than 20 genetic loci have been consistently associated with Alzheimer disease (AD). Accumulating evidence has implicated the endolysosomal pathway in disease pathology. The sortilin-related receptor 1 (SORL1) gene has been associated with AD through multiple studies and has been suggested to function as a guide for amyloid precursor protein (APP) to the endocytic pathway. We identified a family with multiple AD affected individuals that have a single base pair deletion (g.4292delG) resulting in a frameshift alteration and premature termination of the protein (p.Cys143Cys*1). Although previous studies have implicated SORL1 in AD pathogenesis, it remains unclear how this deletion impacts SORL1 protein function in AD development. Patient-specific induced pluripotent stem cells (iPSC) lines were derived to examine the molecular consequences of this SORL1 deletion in neuronal and glial cells (astrocytes and microglia), which can otherwise only be collected from autopsy samples. Each iPSC line generated was validated for pluripotency through immunocytochemical staining and karyotypic stability. Patient iPSC lines and control lines were differentiated into cortical neurons and glia for functional characteristics, including amyloid beta production and uptake, vesicular trafficking, and neurite outgrowth. We have derived iPSC lines bearing the SORL1 deletion and have confirmed pluripotency by immunocytochemistry. These iPSC lines were differentiated into cortical neurons whose identity was confirmed by immunocytochemical staining for neuronal markers. The SORL1 variant bearing cortical neurons showed enlarged endosomes, as measured by RAB5 staining and fluorescent microscopy, compared to the neurons derived from control iPSCs. In addition, the SORL1 deletion bearing neurons showed an upregulation in the ratio of amyloid beta 42:amyloid beta 40 compared to control neurons as measured by ELISA. Finally, SORL1 deletion bearing neurons had reduced neurite outgrowth and branch point numbers compared to controls. In summary, our results support that the p.Cys143Ser*1 variant is pathogenic and alters the functionality of cells from the central nervous system. iPSC-derived neurons and glia cells bearing the deletion in SORL1 p.Cys143Ser*1 variant shows cellular deficits associated with AD pathology, particularly in endolysomal trafficking defects. Studies are continuing to determine if this effect is due to a gain or loss of function.

Theory of Y‐ and Comb‐Shaped Polymer Brushes: The Parabolic Potential Framework

AbstractThe parabolic approximation for self‐consistent molecular potential is widely used for theoretical analysis of conformational and thermodynamic properties of polymer brushes formed by linear or branched macromolecules. The architecture‐dependent parameter of the potential (topological coefficient) can be calculated for arbitrary branched polymer architecture from the condition of elastic stress balance in all the branching points. However, the calculation routine for the topological coefficient does not allow unambiguously identifying the range of applicability and the accuracy of the parabolic approximation. Here the limits of applicability of parabolic approximation are explored by means of numerical self‐consistent field method for brushes formed by Y‐shaped and comb‐like polymers. It is demonstrated that violation of the potential parabolic shape can be evidenced by appearance of multimodal distribution of the end monomer unit in the longest elastic path of the macromolecule. The asymmetry of branching of Y‐shaped polymers does not disturb the parabolic shape of the potential as long as the degree of polymerization of the root segment remains sufficiently large. The same applies to comb‐shape polymers with sufficiently long main chain and large number of branching points. For short comb‐like polymers multiple modes in the distribution of the end monomer unit of the main chain are observed and related to deviation from the parabolic shape of the potential.

Vitamin B12-folic acid supplementation regulates neuronal immediate early gene expression and improves hippocampal dendritic arborization and memory in old male mice

As the relationship among diet, brain aging and memory is complex, it provides ample opportunity for research in multiple directions including behaviour, epigenetics and neuroplasticity. Nutritional deficiencies together with genetic and environmental factors are the major cause of many age-associated pathologies including memory loss. A compromised vitamin B12-folate status in older people is highly prevalent worldwide. Researchers have established a close association between the adequate level of B12-folate and the maintenance of cognitive brain functions. One of the main reasons for age-associated memory loss is downregulation of neuronal immediate early genes (nIEGs). Therefore, we hypothesize here that vitamin B12-folic acid supplementation in old mice can improve memory by altering the expression status of nIEGs. To check this, 72-week-old male Swiss albino mice were orally administered with 2 μg of vitamin B12 and 22 μg of folic acid/mouse/day for eight weeks. Such supplementation improved recognition memory in old and altered the expression of nIEGs. The expression of nIEGs was further found to be regulated by changes in DNA methylation at their promoter regions and CREB phosphorylation (pCREB). In addition, Golgi-Cox staining showed significant improvement in dendritic length, number of branching points and spine density of hippocampal CA1 pyramidal neurons by B12-folic acid supplementation. Taken together, these findings suggest that vitamin B12-folic acid supplementation regulates nIEGs expression and improves dendritic arborization of hippocampal neurons and memory in old male mice.