Cryosection Images Research Articles

Proposing a Methodology for Axon-Centric Analysis of IOP-Induced Mechanical Insult.

IOP-induced mechanical insult on retinal ganglion cell axons within the optic nerve head (ONH) is believed to be a key factor in axonal damage and glaucoma. However, most studies focus on tissue-level mechanical deformations, overlooking that axons are long and thin, and that their susceptibility to damage likely depends on the insult's type (e.g. stretch/compression) and orientation (longitudinal/transverse). We propose an axon-centric approach to quantify IOP-induced mechanical insult from an axon perspective. We used optical coherence tomography (OCT) scans from a healthy monkey eye along with histological images of cryosections to reconstruct the axon-occupied volume including detailed lamina cribrosa (LC) pores. Tissue-level strains were determined experimentally using digital volume correlation from OCT scans at baseline and elevated IOPs, then transformed into axonal strains using axon paths estimated by a fluid mechanics simulation. Axons in the LC and post-LC regions predominantly experienced longitudinal compression and transverse stretch, whereas those in the pre-LC and ONH rim mainly suffered longitudinal stretch and transverse compression. No clear patterns were observed for tissue-level strains. Our approach allowed discerning axonal longitudinal and transverse mechanical insults, which are likely associated with different mechanisms of axonal damage. The technique also enabled quantifying insult along individual axon paths, providing a novel link relating the retinal nerve fiber layer and the optic nerve through the LC via individual axons. This is a promising approach to establish a clearer connection between IOP-induced insult and glaucoma. Further studies should evaluate a larger cohort.

Improving the Signal Intensity of Cryosections Using a Conductive Adhesive Film in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to obtain the molecular images of cryosections without labeling. Although MALDI-MSI has been widely used to detect small molecules from biological tissues, issues remain due to the technical process of cryosectioning and limited mass spectrometry parameters. The use of a conductive adhesive film is a unique method to obtain high-quality sections from cutting tissue, such as bone, muscle, adipose tissue, and whole body of mice or fish, and we have reported the utilization of the film for MALDI-MSI in previous. However, some signal of the small molecules using the conductive adhesive films was still lower than on the indium tin oxide (ITO) glass slide. Here, the sample preparation and analytical conditions for MALDI-MSI using an advanced conductive adhesive film were optimized to obtain strong signals from whole mice heads. The effects of tissue thickness and laser ionization power on signal intensity were verified using MALDI-MSI. The phospholipid signal intensity was measured for samples with three tissue thicknesses (5, 10, and 20 μm); compared to the signals from the samples on the ITO glass slides, the signals with conductive adhesive films exhibited significantly higher intensities when a laser with a higher range of power was used to ionize the small molecules. Thus, the technique using the advanced conductive adhesive film showed an improvement in MALDI-MSI analysis.

Computational study of the mechanical behavior of the astrocyte network and axonal compartments in the mouse optic nerve head.

Glaucoma is a blinding disease characterized by the degeneration of the retinal ganglion cell (RGC) axons at the optic nerve head (ONH). A major risk factor for glaucoma is the intraocular pressure (IOP). However, it is currently impossible to measure the IOP-induced mechanical response of the axons of the ONH. The objective of this study was to develop a computational modeling method to estimate the IOP-induced strains and stresses in the axonal compartments in the mouse astrocytic lamina (AL) of the ONH, and to investigate the effect of the structural features on the mechanical behavior. We developed experimentally informed finite element (FE) models of six mouse ALs to investigate the effect of structure on the strain responses of the astrocyte network and axonal compartments to pressure elevation. The specimen-specific geometries of the FE models were reconstructed from confocal fluorescent images of cryosections of the mouse AL acquired in a previous study that measured the structural features of the astrocytic processes and axonal compartments. The displacement fields obtained from digital volume correlation in prior inflation tests of the mouse AL were used to determine the displacement boundary conditions of the FE models. We then applied Gaussian process regression to analyze the effects of the structural features on the strain outcomes simulated for the axonal compartments. The axonal compartments experienced, on average, 6 times higher maximum principal strain but 1800 times lower maximum principal stress compared to those experienced by the astrocyte processes. The strains experienced by the axonal compartments were most sensitive to variations in the area of the axonal compartments. Larger axonal compartments that were more vertically aligned, closer to the AL center, and with lower local actin area fraction had higher strains. Understanding the factors affecting the deformation in the axonal compartments will provide insights into mechanisms of glaucomatous axonal damage.

Machine learning for cryosection pathology predicts the 2021 WHO classification of glioma

Timely and accurate intraoperative cryosection evaluations remain the gold standard for guiding surgical treatments for gliomas. However, the tissue-freezing process often generates artifacts that make histologic interpretation difficult. In addition, the 2021 WHO Classification of Tumors of the Central Nervous System incorporates molecular profiles in the diagnostic categories, so standard visual evaluation of cryosections alone cannot completely inform diagnoses based on the new classification system. To address these challenges, we develop the context-aware Cryosection Histopathology Assessment and Review Machine (CHARM) using samples from 1,524 glioma patients from three different patient populations to systematically analyze cryosection slides. Our CHARM models successfully identified malignant cells (AUROC= 0.98± 0.01 in the independent validation cohort), distinguished isocitrate dehydrogenase (IDH)-mutant tumors from wild type (AUROC= 0.79-0.82), classified three major types of molecularly defined gliomas (AUROC= 0.88-0.93), and identified the most prevalent subtypes of IDH-mutant tumors (AUROC= 0.89-0.97). CHARM further predicts clinically important genetic alterations in low-grade glioma, including ATRX, TP53, and CIC mutations, CDKN2A/B homozygous deletion, and 1p/19q codeletion via cryosection images. Our approaches accommodate the evolving diagnostic criteria informed by molecular studies, provide real-time clinical decision support, and will democratize accurate cryosection diagnoses. Supported in part by the National Institute of General Medical Sciences grant R35GM142879, the Google Research Scholar Award, the Blavatnik Center for Computational Biomedicine Award, the Partners' Innovation Discovery Grant, and the Schlager Family Award for Early Stage Digital Health Innovations.

CD206+ tendon resident macrophages and their potential crosstalk with fibroblasts and the ECM during tendon growth and maturation.

Resident macrophages exist in a variety of tissues, including tendon, and play context-specific roles in their tissue of residence. In this study, we define the spatiotemporal distribution and phenotypic profile of tendon resident macrophages and their crosstalk with neighboring tendon fibroblasts and the extracellular matrix (ECM) during murine tendon development, growth, and homeostasis. Fluorescent imaging of cryosections revealed that F4/80+ tendon resident macrophages reside adjacent to Col1a1-CFP+ Scx-GFP+ fibroblasts within the tendon fascicle from embryonic development (E15.5) into adulthood (P56). Through flow cytometry and qPCR, we found that these tendon resident macrophages express several well-known macrophage markers, including Adgre1 (F4/80), Mrc1 (CD206), Lyve1, and Folr2, but not Ly-6C, and express the Csf1r-EGFP ("MacGreen") reporter. The proportion of Csf1r-EGFP+ resident macrophages in relation to the total cell number increases markedly during early postnatal growth, while the density of macrophages per mm2 remains constant during this same time frame. Interestingly, proliferation of resident macrophages is higher than adjacent fibroblasts, which likely contributes to this increase in macrophage proportion. The expression profile of tendon resident macrophages also changes with age, with increased pro-inflammatory and anti-inflammatory cytokine expression in P56 compared to P14 macrophages. In addition, the expression profile of limb tendon resident macrophages diverges from that of tail tendon resident macrophages, suggesting differential phenotypes across anatomically and functionally different tendons. As macrophages are known to communicate with adjacent fibroblasts in other tissues, we conducted ligand-receptor analysis and found potential two-way signaling between tendon fibroblasts and resident macrophages. Tendon fibroblasts express high levels of Csf1, which encodes macrophage colony stimulating factor (M-CSF) that acts on the CSF1 receptor (CSF1R) on macrophages. Importantly, Csf1r-expressing resident macrophages preferentially localize to Csf1-expressing fibroblasts, supporting the "nurturing scaffold" model for tendon macrophage patterning. Lastly, we found that tendon resident macrophages express high levels of ECM-related genes, including Mrc1 (mannose receptor), Lyve1 (hyaluronan receptor), Lair1 (type I collagen receptor), Ctss (elastase), and Mmp13 (collagenase), and internalize DQ Collagen in explant cultures. Overall, our study provides insights into the potential roles of tendon resident macrophages in regulating fibroblast phenotype and the ECM during tendon growth.

Multiplexed tape-stabilized cryohistology of mineralized large animal specimens.

Tape-stabilized cryohistology is a powerful histological method to reinforce tissue samples during and after sectioning, enhancing the overall image quality. This technique has widely been applied to section mineralized small animal (i.e., mice, rat, rabbit) specimens, but has only been sparsely implemented for large animal samples that have a greater tendency to tear due to their increased surface area. Here, we present an optimized protocol for tape-stabilized cryohistology of undecalcified minipig vertebral body, femoral head, and temporomandibular joint samples. This protocol further develops a pipeline for sequential staining and imaging of the tape-stabilized cryosections. Images from multiple rounds of staining (endogenous bone mineral labels, aligned collagen (polarized light), tartrate resistant phosphatase (TRAP), alkaline phosphatase (AP), and toluidine blue) are overlaid to provide insight into dynamic bone remodeling. Overall, the established multiplexed tape-stabilized cryohistology protocol provides step-by-step instructions and guidance to cryosection large, mineralized tissues, and maximize data output from a single histological section.

Spatial metabolomics reveals upregulation of several pyrophosphate-producing pathways in cortical bone of Hyp mice.

Patients with the renal phosphate-wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI-based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency-induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.

Tissue distribution and metabolic profiling of cyclosporine (CsA) in mouse and rat investigated by DESI and MALDI mass spectrometry imaging (MSI) of whole-body and single organ cryo-sections.

Therapeutic peptides are a fast-growing class of pharmaceuticals. Like small molecules, the costs associated with their discovery and development are significant. In addition, since the preclinical data guides first-in-human studies, there is a need for analytical techniques that accelerate and improve our understanding of the absorption, distribution, metabolism, and excretion (ADME) characteristics of early drug candidates. Mass spectrometry imaging (MSI), which can be used to visualize drug distribution in intact tissue, has been extensively used to study small molecule drugs, but only applied to a limited extent to larger molecules, such as peptides, after dosing. Herein, we use MSI to obtain spatial information on the distribution and metabolism of a peptide drug. The immunosuppressant cyclosporine (CsA), a cyclic undecapeptide, was used as a-proof-of-concept peptide and investigated by desorption electrospray ionization (DESI) MSI. Calibration curves were made based on a spiked tissue homogenate model. Different washing protocols were tested to improve sensitivity, but CsA, being a quite lipophilic peptide, was found not to benefit from tissue washing. The distribution of CsA and its metabolites were mapped in whole-body mouse sections and within rat organs. Whole-body DESI-MSI studies in mice showed widespread distribution of CsA with highest abundance in organs like the pancreas and liver. After 24h, hydroxy and dihydroxy metabolites of CsA were detected predominantly in the intestines, which were largely devoid of CsA. In addition to the DESI-MSI experiments, MALDI-MSI was also conducted on rat jejunum at higher spatial resolution, revealing the morphology of the jejenum at greater detail; however, DESI provided similar results for drug and metabolite distribution in rat jejunum at apparent slightly better sensitivity. Given its label-free nature, MSI could provide valuable ADME insight, especially for candidates in the early-stage pipeline before radiolabeling.

Super-resolved fluorescence imaging of peripheral nerve

Traditional histopathologic evaluation of peripheral nerve employs brightfield microscopy with diffraction limited resolution of ~ 250 nm. Though electron microscopy yields nanoscale resolution of the nervous system, sample preparation is costly and the technique is incompatible with living samples. Super-resolution microscopy (SRM) comprises a set of imaging techniques that permit nanoscale resolution of fluorescent objects using visible light. The advent of SRM has transformed biomedical science in establishing non-toxic means for investigation of nanoscale cellular structures. Herein, sciatic nerve sections from GFP-variant expressing mice, and regenerating human nerve from cross-facial autografts labelled with a myelin-specific fluorescent dye were imaged by super-resolution radial fluctuation microscopy, stimulated emission depletion microscopy, and structured illumination microscopy. Super-resolution imaging of axial cryosections of murine sciatic nerves yielded robust visualization myelinated and unmyelinated axons. Super-resolution imaging of axial cryosections of human cross-facial nerve grafts demonstrated enhanced resolution of small-caliber thinly-myelinated regenerating motor axons. Resolution and contrast enhancement afforded by super-resolution imaging techniques enables visualization of unmyelinated axons, regenerating axons, cytoskeleton ultrastructure, and neuronal appendages of mammalian peripheral nerves using light microscopes.

True-color 3D rendering of human anatomy using surface-guided color sampling from cadaver cryosection image data: A practical approach.

Three‐dimensional computer graphics are increasingly used for scientific visualization and for communicating anatomical knowledge and data. This study presents a practical method to produce true‐color 3D surface renditions of anatomical structures. The procedure involves extracting the surface geometry of the structure of interest from a stack of cadaver cryosection images, using the extracted surface as a probe to retrieve color information from cryosection data, and mapping sampled colors back onto the surface model to produce a true‐color rendition. Organs and body parts can be rendered separately or in combination to create custom anatomical scenes. By editing the surface probe, structures of interest can be rendered as if they had been previously dissected or prepared for anatomical demonstration. The procedure is highly flexible and nondestructive, offering new opportunities to present and communicate anatomical information and knowledge in a visually realistic manner. The technical procedure is described, including freely available open‐source software tools involved in the production process, and examples of color surface renderings of anatomical structures are provided.

Facial Treatment with 3-O-Cetyl Ascorbic Acid for Improvement of Skin Texture: Uptake, Effectiveness, and In Vitro Carcinogenicity Assessment

Ascorbic acid (AA) is a water-soluble vitamin that is found at high concentrations in normal skin. The important and well-known benefits of using AA in skin health include the stimulation of collagen synthesis and the assistance of protection against photo-oxidative damages. To maintain stability and improve drug delivery to the active site, a variety of AA derivatives have been chemically synthesized. Among these compounds, we focus here on a lipophilic derivative, 3-O-cetyl ascorbic acid (3-CetylAA), which remains poorly characterized for cosmetic applications. Uptake analysis in three healthy human volunteers’ skin was conducted using a serial tape-stripping technique detecting 3-CetylAA (on average, 128 ± 27 pmol per µg) in the stratum corneum after a 5-h topical treatment when treated with 25 mM 3-CetylAA-containing cream for 13 days twice daily and continuously. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging of vertical cryosections of pig skin revealed the presence of 3-CetylAA in the epidermal layer after topical treatment with 3-CetylAA-containing cream. In sun-exposed human skin, 3-CetylAA improved the texture after treatment with 25 mM 3-CetylAA-containing cream for 4 weeks or more when used twice daily or continuously. An in vitro transformation assay using BALB/c 3T3 A31-1-1 cells demonstrated that 10 µM 3-CetylAA, which is the same concentration exhibited in vitro biological activities in another lipophilic AA derivative, 2-O-octadecyl ascorbic acid, was non-carcinogenic and did not potentiate the UVC-induced transformation frequency when applied for 3 days after UVC irradiation. These results demonstrate that 3-CetylAA is a promising candidate as a lipophilic derivative of AA for cosmetic purposes.

Transduction of Mouse Retina By Insect Cell Packaged Recombinant adeno-associated Viruses and Their Mutants Via Intravitreal Injection

Aim: Adeno-associated virus (AAV) is the most preferred gene therapy vector. The purpose of our research is to compare the infection tropism and gene expression efficiency of vitreous injection of recombinant AAVs (rAAVs) and their capsid mutants in mouse retina. Materials & methods: We packaged wild-type rAAV2/2,6,8,9 and their capsid mutants carrying EGFP expression cassette using insect cells. The gene expression profiles of rAAVs and their mutants in mouse retina were evaluated by optical imaging of retinal tissue flat mount and cryosections. Results & conclusion: The results showed that rAAV2 and rAAV2-Y444F mainly targeted retinal ganglion cell; rAAV8, rAAV8-Y733F, rAAV9 and mutants had obvious EGFP expression in retinal pigment epithelium cells. Compared with the wild-type rAAVs, capsid mutants have an improved transduction efficiency in mouse retina cells.

Cross-sectional anatomy, computed tomography, and magnetic resonance imaging of the banded houndshark (Triakis scyllium)

Due to their important phylogenetic position among extant vertebrates, sharks are an invaluable group in evolutionary developmental biology studies. A thorough understanding of shark anatomy is essential to facilitate these studies and documentation of this iconic taxon. With the increasing availability of cross-sectional imaging techniques, the complicated anatomy of both cartilaginous and soft tissues can be analyzed non-invasively, quickly, and accurately. The aim of this study is to provide a detailed anatomical description of the normal banded houndshark (Triakis scyllium) using computed tomography (CT) and magnetic resonance imaging (MRI) along with cryosection images. Three banded houndsharks were scanned using a 64-detector row spiral CT scanner and a 3 T MRI scanner. All images were digitally stored and assessed using open-source Digital Imaging and Communications in Medicine viewer software in the transverse, sagittal, and dorsal dimensions. The banded houndshark cadavers were then cryosectioned at approximately 1-cm intervals. Corresponding transverse cryosection images were chosen to identify the best anatomical correlations for transverse CT and MRI images. The resulting images provided excellent detail of the major anatomical structures of the banded houndshark. The illustrations in the present study could be considered as a useful reference for interpretation of normal and pathological imaging studies of sharks.

Geographic Atrophy: Confocal Scanning Laser Ophthalmoscopy, Histology, and Inflammation in the Region of Expanding Lesions.

PurposeTo describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion.MethodsEnucleated, fixed eyes from seventeen donors with GA were imaged and analyzed by BAF-SLO, IRAF-SLO, and by fundus macroscopy (FM). Tissue from the margins of the GA lesions was cut and processed for resin embedding and histology or cryosectioning and fluorescence in the green and far-red channels, and immunohistochemistry to assess markers of inflammation. Isolated DNA from donors was genotyped for single nucleotide polymorphisms (SNPs) previously shown to be risk factors for the development and progression of AMD.ResultsAround the leading edge of the GA lesions we observed hypertrophic RPE cells with cytoplasm filled with granules fluorescent both in the far-red and green-red channels; abundant microglia and macrophage; deposition of complement factor H (CFH) in Bruch's membrane (BM) and increased membrane attack complex (MAC) on RPE cells.ConclusionsFluorescence imaging of cryosections of RPE cells around the leading edge of the GA lesions suggest that IRAF-SLO visualizes mostly melanin-related compounds. In addition, medium-size GA atrophy displayed the most significant changes in inflammation markers.

Label-free histomorphometry of peripheral nerve by stimulated Raman spectroscopy.

Conventional processing of nerve for histomorphometry is resource-intensive, precluding use in intraoperative assessment of nerve quality during nerve transfer procedures. Stimulated Raman scattering (SRS) microscopy is a label-free technique that enables rapid and high-resolution histology. Segments of healthy murine sciatic nerve, healthy human obturator nerve, and human cross-facial nerve autografts were imaged on a custom SRS microscope. Myelinated axon quantification was performed through segmentation using a random forest machine learning algorithm in commercial software. High contrast, high-resolution imaging of nerve morphology was obtained with SRS imaging. Automated myelinated axon quantification from cross-sections of healthy human nerve imaged using SRS was achieved. Herein, we demonstrate the use of a label-free technique for rapid imaging of murine and human peripheral nerve cryosections. We illustrate the potential of this technique to inform intraoperative decision-making through rapid automated quantification of myelinated axons using a machine learning algorithm.

A novel calibration strategy based on internal standard\u2013spiked gelatine for quantitative bio-imaging by LA-ICP-MS: application to renal localization and quantification of uranium

Mass spectrometry imaging (MSI) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been employed for the elemental bio-distribution and quantification of uranium (U) in histological tissue sections of rodent kidneys. Kidneys were immediately immersed into 4% paraformaldehyde (PFA) solution for 24 h, Tissue-Tek O.C.T. Compound embedded and stored at − 80 °C until cutting in a cryostat, and mounted in gel-covered glass slides. In order to assure complete ablation of sample, sample preparation and laser conditions were carefully optimized. In this work, a new analytical methodology is presented for performing quantitative laser ablation analyses based on internal standard (thulium, Tm)–spiked gelatine (10% m/v) for correction of matrix effects, lack of tissue homogeneity, and instrumental drift. In parallel, matrix-matched laboratory standards, dosed at different concentrations of U, were prepared from a pool of rat kidneys. The quantitative images of cryo-sections revealed heterogeneous distribution of uranium within the renal tissue, because the cortical concentration was up to 120-fold higher than the medullary concentration.Graphical abstract

Anatomy Studio: A tool for virtual dissection through augmented 3D reconstruction

3D reconstruction from anatomical slices allows anatomists to create three dimensional depictions of real structures by tracing organs from sequences of cryosections. However, conventional user interfaces rely on single-user experiences and mouse-based input to create content for education or training purposes. In this work, we present Anatomy Studio, a collaborative Mixed Reality tool for virtual dissection that combines tablets with styli and see-through head-mounted displays to assist anatomists by easing manual tracing and exploring cryosection images. We contribute novel interaction techniques intended to promote spatial understanding and expedite manual segmentation. By using mid-air interactions and interactive surfaces, anatomists can easily access any cryosection and edit contours, while following other user’s contributions. A user study including experienced anatomists and medical professionals, conducted in real working sessions, demonstrates that Anatomy Studio is appropriate and useful for 3D reconstruction. Results indicate that Anatomy Studio encourages closely-coupled collaborations and group discussion, to achieve deeper insights.

Stain-Free Resolution of Unmyelinated Axons in Transgenic Mice Using Fluorescence Microscopy

Though unmyelinated fibers predominate axon counts within peripheral nerves, they are frequently excluded in histomorphometric assessment as they cannot be readily resolved by light microscopy. Herein, we demonstrate stain-free resolution of unmyelinated axons in Sox10-Venus mice by widefield fluorescence imaging of sciatic nerve cryosections. Optional staining of cryosections using a rapid and nontoxic myelin-specific dye (FluoroMyelin Red) enables robust synchronous resolution of myelinated and unmyelinated fibers, comprising a high-throughput platform for neural histomorphometry.

MTORC1 and mTORC2 are differentially engaged in the development of laser-induced CNV

BackgroundThe mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model.MethodsIn experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12 days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development.ResultsIn experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, α-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy.ConclusionOur study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.

3D modelling of non-intestinal colorectal anatomy.

There is a paucity of methods to model soft anatomical tissues. Accurate modelling of these tissues can be difficult with current medical imaging technology. The aim of this research was to develop a methodology to model non-intestinal colorectal tissues that are not readily identifiable radiologically to enhance contextual understanding of these tissues and inform medical device design. The models created were used to inform the design of a novel medical device to separate the mesocolon from the retroperitoneum during resection of the colon. We modelled the peritoneum and the mesentery. The mesentery was used to indicate the location of Toldt's fascia. We generated a point cloud dataset using cryosection images as the target anatomy is more visible than in CT or MRI images. The thickness of the mesentery could not be accurately determined as point cloud data do not have thickness. A denser point cloud detailing the mesenteric boundaries could be used to address this. Expert anatomical and surgical insight and point cloud data modelling methods can be used to model soft tissues. This research enhances the overall understanding of the mesentery and Toldt's fascia in the human specimen which is necessary for medical device innovations for colorectal surgical procedures.