Canalicular System Research Articles

Combined use of serial block face SEM and focused ion beam SEM elucidates the 3D ultrastructure of blood platelets and thrombi

With the emergence of the 3D electron microscopy techniques, serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), it has become feasible to image cellular and tissue ultrastructure at a nanoscale. In the field of homeostasis and thrombosis, both techniques enable investigation of platelet activation and clotting. We find that FIB-SEM provides 3D ultrastructure from volumes in the range 10,000 to 100,000 cubic micrometers, revealing alpha-granules and the canalicular system at a near-isotropic spatial resolution of ∼5 nm.

Quercetin-loaded platelets as a potential targeted therapy for glioblastoma multiforme cell line U373-MG.

Over the globe, the incidence of glioblastoma multiforme (GM) is very low, that is, 1-4 cases per 100,000, but it is fatal and cancer grows very fast inside the brain tissues, namely astrocytes and oligodendrocytes. Because of the rapid growth, it is difficult to halt the dissemination of tumor in adjacent tissues. Although temozolomide (TMZ) is a currently approved standard of care, it develops resistance over the period. Therefore, there is a need to develop a novel drug delivery system. In this work, authors have developed platelets as drug delivery carriers-loaded with quercetin (QCT) for targeting GM. The effect of QCT and QCT-platelet was assessed on the U373-MG cell line. Natural human platelets were used as carriers for drug loading and drug delivery. Platelets possess an open canalicular system that allows the uptake of drug molecules in the platelet cytoplasm. The study showed that the maximum encapsulation efficiency of QCT-platelet was 93.96± 0.12% and the maximum drug release in 24 h was 76.26± 0.13% in-vitro at pH 5.5 that mimics the tumor microenvironment. In this work, there is a three-fold enhancement of solubility of QCT. The cytotoxic activity of QCT-platelets was studied in the U373-MG human astrocytoma glioblastoma cell line and the cell viability was 14.52± 1.53% after 48 h. Thus, platelets were proved as good carriers for therapeutic moieties and can be effectively used to target the glioblastoma tumor in the near future.

Rapid Release of Interleukin-1β from Human Platelets Is Independent of NLRP3 and Caspase.

Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1β (IL-1β), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1β) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1β in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1β. Platelets release IL-1β within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1β in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1β were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1β by a mechanism which is independent of caspase-1 and NLRP3.

Ultrastructural Alterations of Platelets and Monocytes Associated with Autoantibodies in Systemic Lupus Erythematosus

Introduction. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a high titer of autoantibodies, and SLE patients are predisposed to thrombosis. Monocytes and platelets are potentially involved in the pathogenesis of SLE. Monocytes are a source of immune modulators for T and B lymphocytes and they increase the risk of thrombosis through exposing tissue factor on their cell surface. Platelets are activated in SLE and may promote thrombotic events by expressing the procoagulant phosphatidylserine. The aim of this work was to investigate the ultrastructural changes of monocytes and platelets in SLE patients in association with titers of autoantibodies.Methods. SLE patients were included in the study based on the diagnostic criteria of the American College of Rheumatology. Platelets from 5 SLE patients and 3 healthy donors were isolated by gel-filtration on Sepharose 2B from citrated platelet-rich plasma. Monocytes of 6 SLE patients and 3 healthy donors were isolated using density gradient centrifugation of whole citrated blood followed by negative immune-separation. The >96% purity of the isolated monocytes was confirmed using flow cytometry and the viability of the isolated monocytes assessed by exclusion of trypan blue was 95-99%. The ultrastructure of platelets and monocytes from SLE patients and healthy donors was studied with transmission electron microscopy. A total 192 individual platelets and 154 monocytes from SLE patients were analyzed and compared with 60 individual platelets and 22 monocytes from healthy donors.Results . Most platelets from SLE patients and healthy donors had a normal discoid shape without or with 1-2 short filopodia. Intracellular components (α-granules, dense granules, open canalicular system, lysosomes and mitochondria) were well visualized, albeit somewhat less clear in the SLE platelets. The biggest morphological difference between platelets from healthy subjects and SLE patients was revealed in the platelet plasma membrane. All platelets from healthy donors had normal ultrastructure of the plasma membrane, while the membrane of SLE platelets was very often (in 70% of the cells) shaggy and rough and not as smooth as in normal platelets. About 38% of the SLE platelets were fully bordered with a shaggy plasma membrane, while 32% of the platelets had only partially abnormal plasma membrane. Cells from a patient with anti-cardiolipin and anti-beta-2-glypoprotein antibodies had the most striking membrane abnormalities that were found in 100% of platelets analyzed.Monocytes with normal ultrastructure (ruffled cell surface, formation of filopodia, an indented nucleus, and uniform chromatin distribution) comprised 64% of the total in the SLE patients and 95% in the healthy donors. Unlike monocytes from healthy subjects, 19% of monocytes from the SLE patients had ultrastructural signs of apoptosis with a smooth cell surface, electron-dense cytoplasm, mitochondrial swelling, and a spherical nucleus with perinuclear accumulation of condensed chromatin or condensation of chromatin into dense spherical particles. About 17% of monocytes from the SLE patients and 5% from the healthy donors had necrotic features, such as smooth cell surface without or with quite a few filopodia, low electron-density cytoplasm, small electron-dense mitochondria, and spherical pyknotic nucleus with more diffuse euchromatin. The SLE patients with a high titer of anti-dsDNA antibodies (>200 IU/ml) had significantly more apoptotic (25%, p<0.01, χ2-test) and necrotic (20%, p<0.05) monocytes than those of the SLE patients with a normal titer of anti-dsDNA antibodies (<25 IU/ml) (6% and 10%, respectively).Conclusions . In SLE patients, most platelets have abnormal ultrastructure of the plasma membrane (loss of smoothness, shagginess), likely due to deposition of pathological immune complexes. A substantial fraction of monocytes in SLE undergo morphological alternations of apoptotic or necrotic types. Such alterations were more pronounced in patients with an increased level of anti-cardiolipin, anti-beta-2-glypoprotein, and anti-dsDNA autoantibodies. The results confirm the involvement of both platelets and monocytes in the pathogenesis of SLE, including functional and structural alterations associated with increased production of various autoantibodies.The work was supported by the Program for Competitive Growth at Kazan Federal University. DisclosuresNo relevant conflicts of interest to declare.

Super-resolution imaging of platelet-activation process and its quantitative analysis

Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.

Control of Bone Matrix Properties by Osteocytes.

Osteocytes make up 90–95% of the cellular content of bone and form a rich dendritic network with a vastly greater surface area than either osteoblasts or osteoclasts. Osteocytes are well positioned to play a role in bone homeostasis by interacting directly with the matrix; however, the ability for these cells to modify bone matrix remains incompletely understood. With techniques for examining the nano- and microstructure of bone matrix components including hydroxyapatite and type I collagen becoming more widespread, there is great potential to uncover novel roles for the osteocyte in maintaining bone quality. In this review, we begin with an overview of osteocyte biology and the lacunar–canalicular system. Next, we describe recent findings from in vitro models of osteocytes, focusing on the transitions in cellular phenotype as they mature. Finally, we describe historical and current research on matrix alteration by osteocytes in vivo, focusing on the exciting potential for osteocytes to directly form, degrade, and modify the mineral and collagen in their surrounding matrix.

Clinical results of primary repair with silicone tube intubation after traumatic injury of lacrimal canalicular system

Aim: To evaluate clinical results of primary repair with silicone tube intubation after traumatic injury of lacrimal canalicular system.Materials and Methods: Fifteen patients applied to ophthalmology clinic at Afyonkarahisar Health Sciences University betweenJanuary 2010 and May 2020 with traumatic injury of lacrimal canaliculi were included in the study. Data including the patients’demographic characteristics, causes of injury, time between trauma and surgery, surgical technique, post-operative time of siliconetube removal, post-operative follow-up period, and anatomical as well as functional outcomes were retrieved from the patients’clinical records.Results: Mean age of the patients was 26±22.5 (3 to 70 years) years (13 males, 2 females). Ten patients (66.6%) had blunt trauma,whereas penetrating injury was detected as causative factor of trauma in five patients (33.3%). Twelve patients (80%) had isolatedlower canalicular injury, two (13.3%) had upper canalicular injury, and the remaining one (6.7%) had both lower and upper canalicularinjury. The primary surgical repair with silicone tube intubation was performed in all patients. Twelve patients (80%) had successfulanatomical and functional outcomes. Post-operative ptosis was detected in one patient. Although two patients (13.3%) underwentre-operation at another center, status their functional success could not be acquired.Conclusion: Annular intubation with the silicon tube can be used as an effective method for providing anatomical and functionalintegrity of the drainage system after blunt and / or penetrating traumatic injuries of the lacrimal canaliculi.

Evading the host response: Staphylococcus \u201chiding\u201d in cortical bone canalicular system causes increased bacterial burden

Extremity reconstruction surgery is increasingly performed rather than amputation for patients with large-segment pathologic bone loss. Debate persists as to the optimal void filler for this “limb salvage” surgery, whether metal or allograft bone. Clinicians focus on optimizing important functional gains for patients, and the risk of devastating implant infection has been thought to be similar regardless of implant material. Recent insights into infection pathophysiology are challenging this equipoise, however, with both basic science data suggesting a novel mechanism of infection of Staphylococcus aureus (the most common infecting agent) into the host lacunar–canaliculi network, and also clinical data revealing a higher rate of infection of allograft over metal. The current translational study was therefore developed to bridge the gap between these insights in a longitudinal murine model of infection of allograft bone and metal. Real-time Staphylococci infection characteristics were quantified in cortical bone vs metal, and both microarchitecture of host implant and presence of host immune response were assessed. An orders-of-magnitude higher bacterial burden was established in cortical allograft bone over both metal and cancellous bone. The establishment of immune-evading microabscesses was confirmed in both cortical allograft haversian canal and the submicron canaliculi network in an additional model of mouse femur bone infection. These study results reveal a mechanism by which Staphylococci evasion of host immunity is possible, contributing to elevated risks of infection in cortical bone. The presence of this local infection reservoir imparts massive clinical implications that may alter the current paradigm of osteomyelitis and bulk allograft infection treatment.

Platelet Membrane Procoagulation in Preeclampsia

Background: Preeclampsia is a hypertensive disorder in pregnancy that results in significant adverse maternal and neonatal outcomes. Platelet activation is involved in the pathophysiology of preeclampsia and contributes to the prothrombotic state of the disorder. The mechanisms which initiate and sustain platelet activation in preeclampsia, and how platelets are involved in the pathogenesis of coagulation abnormalities in preeclampsia remains unclear.Aim: To utilise a systematic analysis of Procoagulant Membrane Dynamics (PMD) to assess platelet procoagulation in normotensive pregnancies and preeclampsia, compared to age-matched non-pregnant female controls. Methods: Platelets were assessed using 4D live-cell imaging, immunoassay, flow cytometry and impedance aggregometry. Study participants were healthy, non-diabetic, normotensive, non-pregnant controls, (NP, N=9), normotensive pregnant controls (PC, N=9), and pregnant women with preeclampsia (PEE, N=8). Result: Platelets of preeclamptic patients were significantly activated under basal conditions and showed major remodelling of the open canalicular or dense tubular system. Compared to NP and PC controls, PEE platelets showed a marked decrease in dense granule release, and total exposed phosphatidylserine (PS)-laden surface as well as a decreased whole-blood aggregation in response to high concentration collagen stimulation. Notably, in patients who were pregnant, we visualized in both plasma and peripheral whole blood, freely suspended platelet microthrombi ranging from 10-15 µm radius but were more in PEE participants (~75%) than in the normal pregnant controls (~22%). These microthrombi showed fibrin deposits in between procoagulant platelets with ballooned membranes. Unstimulated PEE platelets showed more than a 2-fold increase in the membrane expression of the facultative glucose transporter GLUT3 which normally reside within the intracellular membrane of alpha granules. Multiplex immunoassay for inflammatory markers confirmed the elevation of previously unreported i-309 and C-TACK inflammatory cytokines in PEE participants. Discussion: We elucidated a platelet-based mechanism of procoagulation in patients with preeclampsia; and demonstrated that platelets were highly activated in the basal, resting state in preeclampsia. Platelet hyperactivation in preeclampsia is complex; there is evidence of procoagulant morphologic membrane changes and p-selectin expression, yet platelets were less able to adhere to exposed collagen to form primary hemostatic plugs. Additionally, novel platelet activation and inflammatory markers such as GLUT3, i-309, and C-TACK cytokines requires further study to understand how they mediate the platelet activation process and the pathophysiology of preeclampsia. Conclusion: Platelet hyperactivation and increased GLUT3 membrane expression may contribute to the progression of preeclampsia, and to the thrombotic and coagulopathic maternal complications of the disease. The platelet activation process itself may provide biomarkers for staging and phenotyping preeclampsia and for the prediction of its severity, clinical manifestations, and disease progression. Disclosures Lee: Takeda: Consultancy, Honoraria, Speakers Bureau; Bayer: Honoraria. Poon:Takeda: Other: honoraria for advisory board meeting attendance; Pfizer: Other: honoraria for advisory board meeting attendance; Novo Nordisk: Other: honoraria for advisory board meeting attendance; Roche: Other: honoraria for advisory board meeting attendance; Bayer: Other: honoraria for advisory board meeting attendance, Research Funding; Bioverative/Sanofi: Other: honoraria for advisory board meeting attendance; CSL-Behring: Other: honoraria for advisory board meeting attendance, Research Funding.

Platelet Ultrastructure in Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)

The familial platelet disorder with associated myeloid malignancy (FPDMM) is an autosomal dominant platelet disorder, caused by germline RUNX1 mutations, with predisposition to develop hematologic malignancies, especially acute myeloid leukemia. In many of the FPDMM families reported, the platelet defect was a delta-storage-pool disease (d-SPD) which can also be found without leukemia propensity. However, it has not been studied whether the two types of d-SPD have a common nature. Platelet ultrastructure, previously very little studied, may be one of the aspects to be analyzed to solve this question. We analyzed the ultrastructural characteristics of platelets in 5 members of a family with FPDMM. The family included three generations and all affected members had a RUNX1 deletion: chr21:36349450-36572837 (Rio-Machin et al. Nat Commun 2020;11:1044). None of the patients studied had developed leukemia at the time of the platelet study. We compared the results with those of 24 patients with d-SPD non-associated with leukemia and with those of 15 healthy individuals. Platelets were processed by transmission electron microscopy by standard methods. On the electron micrographs, morphometric analysis of the following structures was performed: 1) Platelets: size and shape, 2) Intraplatelet corpuscular structures (dense granules, alpha granules, mitochondria, lipid droplets): size and number (per platelet and per square micrometer of platelet area), 3) Surface-connected canalicular system (SCS): mean area of individual channel sections and mean percentage of the total SCS area with respect to the platelet area, 4) Glycogen masses: total area with respect to the platelet area. The morphological traits of the platelets and organelles measured above were also evaluated and the dense granules were classified into 4 different types depending on the appearance of their solid core (Weiss et al. Br J Haematol 1993;83:282). The dense tubular system and other ultrastructural characteristics were evaluated by morphology only. The main features of the platelet ultrastructure in patients with FPDMM were (Fig 1, Table 1): 1) slight increase in platelet size while preserving the discoidal shape, 2) moderate reduction in the number of dense granules, which showed a reduced proportion of type 1 granules (with the solid core occupying more than 50 % of the granule) and an increased proportion of type 2 and type 3 granules, with a solid core reduced or fragmented respectively, 3) marked increase and dilatation of SCS with some elements filled by a substance of unknown origin, 4) moderate increase in dense tubular system with occasional complex formation. The platelet ultrastructure was similar to that described in the non-associated d-SPD group (Pujol-Moix et al. Haematologica 2000;85:619) although there were some differences (Table 1): in FPDMM platelets the dense granules were less reduced but more dysmorphic, and the SCS, equally dilated, contained a substance that was not observed in the d-SPD platelets. Given that all the findings described belong to the same family, it would be necessary to evaluate the platelet ultrastructure in additional families and extend the study to other characteristics of d-SPD. Only in this way, it would be possible to know to what extent the platelet defect of FPDMM and that of non-associated d-SPD shared a pathogenic mechanism. Disclosures No relevant conflicts of interest to declare.

Controlling human platelet activation with calcium-binding nanoparticles

Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration. Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation and prevent the unwanted blood clots that causes heart attack and strokes. Previously we have predicted that calcium accumulation within the lumen of an infolded portion of the platelet plasma membrane called the open canalicular system (OCS) is essential for maintaining this cytosolic calcium rise. Due to its nanometer dimensions of the OCS, it has been difficult to measure or interfere with the predicted luminal calcium accumulation. Here we utilise iron oxide magnetic nanoparticles coated with the known calcium chelator, citrate, to create calcium-binding nanoparticles. These were used to assess whether an OCS calcium store plays a role in controlling the dynamics of human platelet activation and aggregation. We demonstrate that citrate-coated nanoparticles are rapidly and selectively uptaken into the OCS of activated human platelets, where they act to buffer the accumulation of calcium there. Treatment with these calcium-binding nanoparticles reduced thrombin-evoked cytosolic calcium rises, and slowed platelet aggregation and clot retraction in human platelets. In contrast, nanoparticles that cannot bind calcium have no effect. This study demonstrates that the OCS acts as a key source of calcium for maintaining cytosolic calcium rises and accelerating platelet aggregation, and that calcium-binding nanoparticles targeted to the OCS could provide an anti-platelet therapy to treat patients at risk of suffering heart attacks or strokes.

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation

Human platelets use a rise in cytosolic Ca2+ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca2+ signals to trigger each of these independently is unknown. Other cells create local Ca2+ signals to activate Ca2+-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca2+ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca2+ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca2+ to isolate a cytosolic Ca2+ nanodomain created by Ca2+ release from the DTS. In the absence of any detectable rise in global cytosolic Ca2+ concentration, thrombin stimulation triggered Na+/Ca2+ exchanger (NCX)-dependent Ca2+ removal into the extracellular space, as well as Ca2+-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca2+ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca2+ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca2+ nanodomain contained within the MC that can control shape change independently of platelet aggregation.

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease

There are few invitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

Results of Traumatic Canalicular Laceration Repair Performed by Trainees in a Tertiary Eye Care Center

Background: Lacrimal canalicular laceration is a common condition in emergency medicine. Many eyelid lacerations involve the lacrimal canalicular system. In the authors’ institution, nearly all cases of lacrimal canalicular laceration are treated by ophthalmology trainees, and are followed-up periodically in the post-operative period. Objective: To analyze the success rate of the cases of lacrimal canalicular laceration treated by trainees in a tertiary eye care center in Thailand. Materials and Methods: Thirty patients with canalicular laceration were treated between January 2005 and December 2017. Data gathered from ICD10, operation records, and in-patient and out-patient medical records were retrospectively analyzed. Results: There were 20 males (66%) and 10 females (34%) with a mean age of 30.86 years (range 1 to 78 years). Seventy percent of patients presented outside of normal office hours. Most injuries were caused by non-vehicular accidents. Twentyfive of 30 cases (83.33%) were repaired by trainees, and 63% of cases were repaired after office hours. The mean waiting time from presentation to repair was 9 hours 56 minutes (range: 3 hours 35 minutes to 22 hours 10 minutes). The follow-up rate at six months postoperative was 53.33%. The success rate of repairs performed by trainees was 93.75%. Conclusion: The success rate of lacrimal canalicular laceration repair performed by trainees was very satisfactory. The average waiting time for surgery was acceptable. Only half of the patients were followed-up for six months post-operative. Improvement is required in the recording of pertinent details in the medical records, which is an important issue to train the trainees. Keywords: Tear canaliculus, Lacrimal canalicular laceration, Repair, Residents, Trainees, Oculoplastic surgeon

Platelets in Amyloidogenic Mice Are Activated and Invade the Brain.

Alzheimer’s disease (AD) is a neurodegenerative disease with a complex and not fully understood pathogenesis. Besides brain-intrinsic hallmarks such as abnormal deposition of harmful proteins, i.e., amyloid beta in plaques and hyperphosphorylated Tau in neurofibrillary tangles, blood-derived elements, in particular, platelets have been discussed to be involved in AD pathogenesis. The underlying mechanisms, however, are rather unexplored. Here, we investigate a potential role of platelets in an AD transgenic animal model with severe amyloid plaque formation, the APP-PS1 transgenic mice, and analyzed the presence, spatial location and activation status of platelets within the brain. In APP-PS1 mice, a higher number of platelets were located within the brain parenchyma, i.e., outside the cerebral blood vessels compared to WT controls. Such platelets were activated according to the expression of the platelet activation marker CD62P and to morphological hallmarks such as membrane protrusions. In the brain, platelets were in close contact exclusively with astrocytes suggesting an interaction between these two cell types. In the bloodstream, although the percentage of activated platelets did not differ between transgenic and age-matched control animals, APP-PS1 blood-derived platelets showed remarkable ultrastructural peculiarities in platelet-specific organelles such as the open canalicular system (OCS). This work urges for further investigations on platelets and their yet unknown functional roles in the brain, which might go beyond AD pathogenesis and be relevant for various age-related neurodegenerative diseases.

Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis

The canalicular system (CS) has been defined as: 1) an inward, invaginated membrane connector that supports entry into and exit from the platelet; 2) a static structure stable during platelet isolation; and 3) the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3. Qualitative and quantitative analysis of whole platelet reconstructions, obtained from immediately fixed or washed platelets fixed post-washing, indicated that CS, even in the presence of activation inhibitors, reorganized during platelet isolation to generate a more interconnected network. Further, CS redistribution into the PM at different times, post-activation, appeared to account for only about half the PM expansion seen in thrombin-activated platelets, in vitro, suggesting that CS reorganization is not sufficient to serve as a dominant membrane reservoir for activated platelets. In sum, our analysis highlights the need to revisit past assumptions about the platelet CS to better understand how this membrane system contributes to platelet function.

Refined fabrication of mechano-stimulating micro-platform for on-chip analyses of complex platelet behavior

Platelets can sense their surroundings as they crawl in search of optimal surface to adhere before activation, but the detailed mechanism has not been fully explored in different microenvironments. Herein, various aspects of platelet behavior, including morphology, movement and biomarker expression, were multilaterally examined on mechanically stimulating surfaces, with controlled rigidity and roughness, to elucidate the effect of microenvironment without chemical stimulus. The platelets demonstrated different movement behavior (e.g. back-and-forth and forward crawling), and motility, as well as varying degrees of spreading with lamellipodial and filopodial projections, based on varying mechanical microenvironments. Furthermore, after initial activation, the platelets all became stationary and the cell spreading became more prominent, while still maintaining their movement potential. P-selectin expression, along with α-granule and open canalicular system (OCS) distributions, all supported the morphological and movement behavior. These results strongly indicated that the mechanical microenvironment played a key role in the regulation of complex platelet activities.

3D ultrastructural analysis of α‐granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets

BackgroundState‐of‐the‐art 3‐dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure. ObjectivesOur goal was to determine the 3D organization of α‐granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships. MethodsWe used serial block face–scanning electron microscopy images to render the 3D ultrastructure of α‐granules, dense granules, mitochondria, canalicular system, and plasma membrane for 30 human platelets, 10 each from 3 donors. α‐Granule compositional data were assessed by sequential, serial section cryo‐immunogold electron microscopy and by immunofluorescence (structured illumination microscopy). Results and Conclusionsα‐Granule number correlated linearly with platelet size, while dense granule and mitochondria number had little correlation with platelet size. For all subcellular compartments, individual organelle parameters varied considerably and organelle volume fraction had little correlation with platelet size. Three‐dimensional data from 30 platelets indicated only limited spatial intermixing of the different organelle classes. Interestingly, almost 70% of α‐granules came within ≤35 nm of each other, a distance associated in other cell systems with protein‐mediated contact sites. Size and shape analysis of the 1488 α‐granules analyzed revealed no more variation than that expected for a Gaussian distribution. Protein distribution data indicated that all α‐granules likely contained the same major set of proteins, albeit at varying amounts and varying distribution within the granule matrix.

Annular intubation with pigtail probe for canalicular lacerations.

Purpose:Pigtail probe as a procedure of choice for canalicular laceration.Methods:Retrospective, consecutive interventional case series of patients with eyelid laceration involving the canaliculus undergoing repair. All patients were subjected to repair with pigtail probe as first choice and only if this was not possible, they were repaired by other method. Outcome was analyzed in terms of cosmetic, functional, and anatomic success.Results:A total of 35 patients (mean age, 10.88 years) underwent eyelid and canaliculus repair by a single surgeon (RM). In all, 32 (91.42%) canalicular lacerations were repaired by annular intubation using a pigtail probe, while the remaining 3 (8.58%) lacerations in which pigtail probe intubation was not possible, were repaired by an alternative method. Upper canaliculus was involved in 6 (17.14%) and lower canaliculus in 29 (82.86%) eyes. Mean follow-up was 8.2 months (range 3–13 months). Intubation tubes were removed after at least 3 months (range 12–20 weeks). None of the patients had complaints of epiphora. All the patients had good cosmesis, anatomic alignment and functional success as assessed by dye disappearance test in younger children and lacrimal irrigation in older children and adults.Conclusion:Bicanalicular annular repair with pigtail probe achieved excellent functional and cosmetic results. The loop minimizes the chances of extrusion of the tube, maintains natural anatomic alignment of the cut ends of the canaliculus and thus retains the integrity of the delicate canalicular system. Pigtail probe intubation can be considered as the first choice in canalicular lacerations.

Selective patterning of netrin-1 as a novel guiding cue for anisotropic dendrogenesis in osteocytes.

Although protein patterning approaches have found widespread applications in tuning surface characteristics of biomaterials, selective control of growth in cell body and dendrites utilizing such platforms remains difficult. The functional roles assumed by cell body and dendrites in a physiological milieu have extremely high specificity. In particular, osteocytes embedded inside the mineralized bone matrix are interconnected via dendritic cell processes characterized by an anisotropic arrangement of the lacunar-canalicular system, where the fluid-flow inside the canaliculi system regulates the mechanoresponsive functionalization of bone. Control of cellular networks connected by dendritic cell processes is, therefore, imperative for constructing artificially controlled bone-mimetic structures and as an extension, for gaining insights into the molecular mechanisms underlying dendrogenesis inside the mineralized bone matrix. Here, we report an innovative strategy to induce controlled elongation of cell body or dendritic process structures in selective directions by using the inkjet printing technique. Artificial runways employing netrin-1, inspired by neural architecture, were utilized to trigger controlled elongation in the osteocyte dendritic processes in desired directions. This is the first report, to the best of our knowledge, demonstrating that anisotropic dendrogenesis of osteocytes can be controlled with selective patterning of extracellular proteins, specifically via the axon guidance ligand netrin-1.