Preclinical in vitro and in vivo results of the new silk vista flow diverter with P8RI coating.

BACKGROUND: Flow diverting stents (FDS) have transformed the treatment of intracranial aneurysms; however, their metallic structure associated with their intra-luminal positioning hamper angiographic and clinical outcomes. Therefore, there is a need to develop FDS with optimized surfaces that reduce thrombogenicity while promoting the healing process and endothelialization. METHODS: P8RI, a peptide mimicking the CD31 protein, was previously developed and grafted onto Silk Vista (SV) FDS. P8RI-SV and bare-SV were used in vitro in a blood loop model to test their hemocompatibility using human whole blood and in vivo using the rabbit elastase model for optical coherence tomography (OCT) comparisons of neointimal formation at day 5 and day 28. RESULTS: After blood loop incubation, P8RI-SV showed significant reduction in fibrin binding (p=0.004) and platelet adhesion (p=0.041) compared with bare-SV. Similarly, derivative markers measured in blood, thromboxane B2 (platelet activation) and Thrombin-Antithrombin III complexes (coagulation activation), were also significantly reduced in the P8RI-SV group (both p=0.002). In vivo, complete or near-complete occlusion was reached in all aneurysms (n=6) at day 28. Excellent rate of stent-coverage ratio was obtained at day 5 (89.3% (79.1%-98.7%)) comparable to the observation at day 28 (91.8% (79.1%-100%); p=0.44). These rates were significantly higher compared with bare-SV at day 5 (77.8% (58.3%-86.8%); p<0.001) and at day 28 (67.7% (52.6%-88.9%); p<0.0001). CONCLUSION: In vitro results confirm enhanced hemocompatibility with a significant anti-thrombotic effect of the P8RI-SV. In vivo results provide evidence of rapid neo-intimal growth reaching near-complete tissue healing as early as day 5 in a rabbit model.

WEB shape modifications: angiography-histopathology correlations in rabbits.

BACKGROUND: WEB Shape Modification (WSM) over time is frequent after aneurysm treatment. In this study, we explored the relationship between histopathological changes and angiographic evolution over time in experimental aneurysms in rabbits treated with the Woven EndoBridge (WEB) procedure. METHODS: Quantitative WSM was assessed using flat-panel computed tomography (FPCT) during follow-up by calculating height and width ratio (HR, WR), defined as the ratio between either measurement at an index time point and the measurement immediately after WEB implantation. The index time point varied from 1 day to 6 months. HR and WR were evaluated with angiographic and histopathological assessments of aneurysm healing. RESULTS: Final HR of devices varied from 0.30 to 1.02 and final WR varied from 0.62 to 1.59. Altogether, at least 5% of HR and WR variations were observed in 37/40 (92.5%) and 28/40 (70%) WEB devices, respectively, at the time of final assessment. There was no significant correlation between complete or incomplete occlusion groups and HR or WR (p=0.15 and p=0.43). Histopathological analysis revealed a significant association between WR and aneurysm healing and fibrosis 1 month following aneurysm treatment (both p<0.05). CONCLUSION: Using longitudinal FPCT assessment, we observed that WSM affects both the height and width of the WEB device. No significant association was found between WSM and aneurysm occlusion status. Although presumably a multifactorial phenomenon, the histopathological analysis highlighted a significant association between width variations, aneurysm healing and fibrosis in the first month following aneurysm treatment.

Comparison of arterial wall integration of different flow diverters in rabbits: The CICAFLOW study.

BACKGROUND AND PURPOSE: New coated flow diverters (FDs) claim antithrombotic properties and increased arterial wall integration. The aim of this study is to compare in vivo endothelial coverage of coated and uncoated FD in the context of different antiplatelet regimens. METHODS: Different FDs (Silk Vista - SV, Pipeline with Shield technology - PED shield and Surpass Evolve - SE) were implanted in the aorta of rabbits, all 3 in each animal with 3 different antiplatelet regimens: no antiplatelet therapy, aspirin alone, or aspirin and ticagrelor. Four weeks after FD implantation, angiography, flat-panel CT, and optical coherence tomography (OCT) were performed before harvesting the aorta. Extensive histopathology analyses were performed including environmental scanning electron microscopy (ESEM), multiphoton microscopy (MPM) and histological staining with qualitative and/or quantitative assessment of device coverage. RESULTS: All 23 FDs that were implanted remained patent without hyperplasia. Qualitative stent coverage assessment revealed that there were no statistically significant differences between the FD groups (p = 0.19, p = 0.45, p = 0.40, and p = 0.84 for OCT, ESEM, MPM and histology, respectively). Quantitative neointimal measurement of histological sections also showed similar results in all 3 FD groups (p = 0.70). However, there were significant differences between the 3 groups of antiplatelet regimens (p = 0.07) with a higher rate in the no antiplatelet group (p = 0.05 versus aspirin alone and p = 0.03 versus aspirin and ticagrelor). CONCLUSION: Our study provides evidence that FD integration into the arterial wall is similar with coated (PED shield) and uncoated devices (SV, SE), regardless of the antiplatelet regimen. FD integration with specific surface coverage should be promoted. TRIAL REGISTRATION: APAFIS #2022011215518538.

Fucoidan-coated coils improve healing in a rabbit elastase aneurysm model.

BACKGROUND: Recanalization of coiled aneurysms remains unresolved. To limit aneurysm recanalization after embolization with coils, we propose an innovative approach to optimize aneurysm healing using fucoidan-coated coils. OBJECTIVE: To evaluate the short-term efficacy and long-term safety of the new coil system with conventional angiography, histology, and multiphoton microscopy for follow-up of fibrosis and neointima formation. METHODS: We conducted a feasibility study on rabbit elastase-induced aneurysms. Embolization was carried out with bare platinum coils, fucoidan-coated coils, or dextran-coated coils. Aneurysms were controlled after 1 month by digital subtraction angiography (DSA). Aneurysm samples were collected and processed for histological analysis. Aneurysm healing and fibrosis were measured by quantifying collagen according to the histological healing score by combining standard light microscopy and multiphoton imaging. We divided 27 rabbits into three groups: bare platinum group, fucoidan group, and dextran group as controls. RESULTS: Angiographic grading showed a trend toward less recanalization in the fucoidan group, although there were no significant differences among the three groups (P=0.21). Histological healing was significantly different according to the presence of more collagen in the neck area of aneurysms in the fucoidan group versus the bare platinum group (P=0.011), but not in the dextran group. Histological index was significantly better at the aneurysm neck in the fucoidan group than in the bare platinum group (P=0.004). Collagen organization index was also significantly better in the fucoidan group than in the bare platinum group (P=0.007). CONCLUSION: This proof-of-concept study demonstrated the feasibility and efficacy of treatment with fucoidan-coated coils to improve aneurysm healing. The results in this rabbit in vivo model showed that fucoidan-coated coils have the potential to improve healing following endovascular treatment.

A novel histological occlusion classification for coiled aneurysms based on multiphoton microscopy.

OBJECTIVE: Intracranial aneurysm (IA) coiling remains the most commonly used endovascular approach for ruptured and unruptured IA, and recanalization is a common drawback that impairs treatment success. Angiographic occlusion and aneurysm healing are not synonymous, and histological evaluation of embolized aneurysms remains a challenge. We propose here an experimental study of coil embolization in animal models by multiphoton microscopy (MPM) in comparison with conventional histological staining. The purpose of his work is to analyze coil healing process using histological sections of aneurysms. METHODS: Based on a rabbit elastase model, 27 aneurysms were fixed, embedded in resin, and cut in thin histological sections 1 month after coils implantation and after angiographic control. Hematoxylin and eosin (H&S) staining were realized. Non-stained adjacent slices were imaged for multiphoton excited autofluorescence (AF) and second-harmonic generation (SHG) to construct three-dimensional (3D) projections of sequentially and axially acquired images. RESULTS: The contrast provided by the combination of these two imaging modalities can be used to distinguish five levels of aneurysm healing, based on a combination of thrombus evolution and increased extracellular matrix (ECM) deposit. CONCLUSION: RDPC:\Users\SHAHUL\RDP6|We have established a novel histological scale from a rabbit elastase aneurysm model after coiling with a classification of five different stages thanks to nonlinear microscopy. This classification is an actualized tool in order to obtain a more precise evaluation of occlusion device efficacy in the scope of new innovative microscopy for research.

Effect of 5-Fluoro-Uracile + Oxaliplatin chemotherapy on the histological response of PEritoneal and hePatIc corectal metasTases in a mOuse model: PEPITO experimental study.

BACKGROUND: The histological responses (HRs) after systemic chemotherapy should be used to determine the optimal management of patients with peritoneal and liver metastasis from colorectal cancer (cPM, cLM), in curative intent. We aimed to compare HRs of cPM and cLM in metastatic mice model after chemotherapy. METHODS: Colon carcinoma CT26-luc cells were transplanted into syngeneic BALB/c mice by intraperitoneal (leading to cPM), intrasplenic (leading to cLM), or intraperitoneal + intrasplenic (leading to cPM cLM) injections and follow up using bioluminescence during 21 days. Bi-chemotherapeutic treatment (5-fluorouracil at D11, D17, and D20, and oxaliplatin at D13 and D19) was administered. The peritoneal cancer index (PCI) and HRs using Peritoneal Regression Grading Score (PRGS) and Tumor Regression Grade (TRG) classifications were analyzed at day 21. RESULTS: Unlike bioluminescence rate, PCI was reduced after chemotherapy in all treated groups with cPM comparatively to controls (33 ± 9.5 vs. 19.8 ± 5, p = 0.002 for cPM groups; 37.7 ± 3.6 vs. 25.2 ± 10.8, p = 0.0003 for the cPM + cLM groups). The complete or major HR rates were higher in all treated groups compared to the non-treated mice (cPM, 2.29 ± 0.55 vs. 3.56 ± 1.01; cLM, 2.43 ± 1.89 vs. 4.86 ± 0.378; cPM + cLM, 2.73 ± 1.03 and 2.2 ± 0.65 vs. 3.79 ± 0.75 and 4.36 ± 0.43). The complete or major HR rates after chemotherapy were similar across the metastatic sites in 60% for cPM + cLM group. CONCLUSIONS: The efficacy of chemotherapeutic treatment did not differ between the metastatic sites. Murine models are suitable in histological analyses to study tumor development and regression but clinical study will be performed to confirm these results.

Feasibility and Safety of Oxaliplatin-Based Pressurized Intraperitoneal Aerosol Chemotherapy With or Without Intraoperative Intravenous 5-Fluorouracil and Leucovorin for Colorectal Peritoneal Metastases: A Multicenter Comparative Cohort Study.

BACKGROUND: This retrospective multicenter cohort study compared the feasibility and safety of oxaliplatin-based pressurized intraperitoneal aerosol chemotherapy (PIPAC-Ox) with or without intraoperative intravenous 5-fluorouracil (5-FU) and leucovorin (L). METHODS: Our study included consecutive patients with histologically proven unresectable and isolated colorectal peritoneal metastases (cPM) treated with PIPAC-Ox in seven tertiary referral centers between January 2015 and April 2020. Toxicity events and oncological outcomes (histological response, progression-free survival, and overall survival) were compared between patients who received intraoperative intravenous 5-FU/L (PIPAC-Ox + 5-FU/L group) and patients who did not (PIPAC-Ox group). RESULTS: In total, 101 patients (263 procedures) were included in the PIPAC-Ox group and 30 patients (80 procedures) were included in the PIPAC-Ox + 5-FU/L group. Common Terminology Criteria for Adverse Events v4.0 grade 2 or higher adverse events occurred in 48 of 101 (47.5%) patients in the PIPAC-Ox group and in 13 of 30 (43.3%) patients in the PIPAC-Ox + 5-FU/L group (p = 0.73). The complete histological response rates according to the peritoneal regression grading score were 27% for the PIPAC-Ox + 5-FU/L group and 18% for the PIPAC-Ox group (p = 0.74). No statistically significant differences were observed in overall or progression-free survival between the two groups. CONCLUSIONS: The safety and feasibility of PIPAC-Ox + 5-FU/L appears to be similar to the safety and feasibility of PIPAC-Ox alone in patients with unresectable cPM. Oncological outcomes must be evaluated in larger studies.

Bioactive refinement for endosaccular treatment of intracranial aneurysms.

Endovascular treatment is the first-line therapy for most intracranial aneurysms; however, recanalisation remains a major limitation. Developments in bioengineering and material science have led to a novel generation of coil technologies for aneurysm embolisation that address clinical challenges of aneurysm recurrence. This review presents an overview of modified surface coil technologies and summarises the state of the art regarding their efficacy and limitations based on experimental and clinical results. We also present potential perspectives to develop biologically optimised devices.

CD31 Mimetic Coating Enhances Flow Diverting Stent Integration into the Arterial Wall Promoting Aneurysm Healing.

BACKGROUND AND PURPOSE: Beyond aneurysmal occlusion, metallic flow diverters (FDs) can induce an adverse endovascular reaction due to the foreignness of metal devices, hampering FD endothelialization across the aneurysm neck, and arterial healing of intracranial aneurysms. Here, we evaluated the potential benefits of an FD coating mimicking CD31, a coreceptor critically involved in endothelial function and endovascular homeostasis, on the endothelialization of FDs implanted in vivo. METHODS: Nitinol FD (Silk Vista Baby) and flat disks were dip-coated with a CD31-mimetic peptide via an intermediate layer of polydopamine. Disks were used to assess the reaction of endothelial cells and blood elements in vitro. An aneurysm rabbit model was used to compare in vivo effects on the arterial wall of CD31-mimetic-coated (CD31-mimetic, n=6), polydopamine-coated (polydopamine, n=6), and uncoated FDs (bare, n=5) at 4 weeks post-FD implantation. In addition, long-term safety was assessed at 12 weeks. RESULTS: In vitro, CD31-mimetic coated disks displayed reduced adhesion of blood elements while favoring endothelial cell attachment and confluence, compared to bare and polydopamine disks. Strikingly, in vivo, the neoarterial wall formed over the CD31-mimetic-FD struts at the aneurysm neck was characteristic of an arterial tunica media, with continuous differentiated endothelium covering a significantly thicker layer of collagen and smooth muscle cells as compared to the controls. The rates of angiographic complete occlusion and covered branch arterial patency were similar in all 3 groups. CONCLUSIONS: CD31-mimetic coating favors the colonization of metallic endovascular devices with endothelial cells displaying a physiological phenotype while preventing the adhesion of platelets and leukocytes. These biological properties lead to a rapid and improved endothelialization of the neoarterial wall at the aneurysm neck. CD31-mimetic coating could therefore represent a valuable strategy for FD biocompatibility improvement and aneurysm healing.

How cellulose nanofibrils and cellulose microparticles impact paper strength-A visualization approach.

Cellulosic nanomaterials are in the focus of academia and industry to realize light-weight biobased materials with remarkable strength. While the effect is well known, the distribution of these nanomaterials are less explored, particularly for paper sheets. Here, we explore the 3D distribution of micro and nanosized cellulosic particles in paper sheets and correlate their extent of fibrillation to the distribution inside the sheets and subsequently to paper properties. To overcome challenges with contrast between the particles and the matrix, we attached probes on the cellulose nano/microparticles, either by covalent attachment of fluorescent dyes or by physical deposition of cobalt ferrite nanoparticles. The increased contrast enabled visualization of the micro and nanosized particles inside the paper matrix using multiphoton microscopy, X-ray microtomography and SEM-EDX. The results indicate that fibrillary fines enrich at pores and fiber-fiber junctions, thereby increasing the relative bonded area between fibers to enhance paper strength while CNF seems to additionally form an inner 3D network.

Multiphoton microscopy for pre-clinical evaluation of flow-diverter stents for treating aneurysms.

BACKGROUND: Conventional histological analyses are the gold standard for the study of aneurysms and vascular pathologies in pre-clinical research. Over the past decade, in vivo and ex vivo imaging using multiphoton microscopy have emerged as powerful pre-clinical tools for detailed tissue analyses that can assess morphology, the extracellular matrix (ECM), cell density and vascularisation. Multiphoton microscopy allows for deeper tissue penetration with minor phototoxicity. OBJECTIVE: The present study aimed to demonstrate the current status of multimodality imaging, including multiphoton microscopy, for detailed analyses of neo-endothelialisation and ECM evolution after flow-diverter stent (FDS) treatment in an experimental rabbit model of aneurysms. METHODS: Multiphoton microscopy tools for assessing autofluorescence and second harmonic generation (SHG) signals from biological tissues were used to evaluate the endovascular treatment of intracranial aneurysms in an animal model of aneurysms (pig, rabbit). Results from multiphoton microscopy were compared to those from standard histology, electronic and bright field microscopy. CONCLUSIONS: The present study describes novel evaluation modes based on multiphoton microscopy for visualising tissue morphology (e.g., collagen, elastin, and cells) to qualify and quantify the extent of neo-intimal formation of covered arteries and device integration into the arterial wall using a rabbit model of intracranial aneurysms treated with FDS.

Long-term survival after surgery of pancreatic primary squamous cell carcinoma: A case report and literature review.

Pancreatic primary squamous cell carcinoma is rarer and no optimal treatment has been validated according to the tumor stage. The surgical resection was the only curative option. The radiotherapy or chemotherapy was performed for the other cases.

Comparison of implantation sites for the development of peritoneal metastasis in a colorectal cancer mouse model using non-invasive bioluminescence imaging.

The development of cancer mouse models is still needed for the identification and preclinical validation of novel therapeutic targets in colorectal cancer, which is the third leading cause of cancer-related deaths in Europe. The purpose of this study was to determine the most accurate tumour cell injection method to obtain suitable peritoneal metastasis (PM) for subsequent therapeutic treatments. Here, we grafted murine colon carcinoma CT-26 cells expressing luciferase into immunocompetent BALB-c mice by intravenous injection (IV group), subcutaneous injection (SC group), intraperitoneal injection after peritoneal scratching (A group) or intraperitoneal injection alone (IP group). Tumour growth was monitored by bioluminescence during the first 15 days post-grafting. The peritoneal carcinomatosis index was evaluated macroscopically, histology, immunohistochemistry and multiphoton microscopy were performed in peritoneal tumour tissue. Upon implantation, no tumour growth was observed in the IV group, similar to the non-injected group. Both the IP and SC groups showed intermediate growth rates, but the SC group produced only a single subcutaneous nodule. The A group exhibited the highest tumour growth at 15 days post-surgery. Anatomic and histologic analyses corroborated the existence of various tumour nodules, and multiphoton microscopy was used to evaluate tumour fibrosis-infiltrating cells in a non-pathologic peritoneum. In conclusion, limited PM was obtained by IP injection, whereas IP injection after peritoneal scratching led to an extensive PM murine model for evaluating new therapeutics.

A readily usable two-photon fluorescence lifetime microendoscope.

A two-photon fluorescence lifetime (2P-FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub-cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table-top microscope performances are achieved through a comprehensive system including a home-designed spectro-temporal pulse shaper and a custom air-silica double-clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 µm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.

Plasma membrane depolarization and permeabilization due to electric pulses in cell lines of different excitability.

In electroporation-based medical treatments, excitable tissues are treated, either intentionally (irreversible electroporation of brain cancer, gene electrotransfer or ablation of the heart muscle, gene electrotransfer of skeletal muscles), or unintentionally (excitable tissues near the target area). We investigated how excitable and non-excitable cells respond to electric pulses, and if electroporation could be an effective treatment of the tumours of the central nervous system. For three non-excitable and one excitable cell line, we determined a strength-duration curve for a single pulse of 10ns-10ms. The threshold for depolarization decreased with longer pulses and was higher for excitable cells. We modelled the response with the Lapicque curve and the Hodgkin-Huxley model. At 1µs a plateau of excitability was reached which could explain why high-frequency irreversible electroporation (H-FIRE) electroporates but does not excite cells. We exposed cells to standard electrochemotherapy parameters (8×100µs pulses, 1Hz, different voltages). Cells behaved similarly which indicates that electroporation most probably occurs at the level of lipid bilayer, independently of the voltage-gated channels. These results could be used for optimization of electric pulses to achieve maximal permeabilization and minimal excitation/pain sensation. In the future, it should be established whether the in vitro depolarization correlates to nerve/muscle stimulation and pain sensation in vivo.

Fast epi-detected broadband multiplex CARS and SHG imaging of mouse skull cells.

We present a bimodal imaging system able to obtain epi-detected mutiplex coherent anti-Stokes Raman scattering (M-CARS) and second harmonic generation (SHG) signals coming from biological samples. We studied a fragment of mouse parietal bone and could detect broadband anti-Stokes and SHG responses originating from bone cells and collagen respectively. In addition we compared two post-processing methods to retrieve the imaginary part of the third-order nonlinear susceptibility related to the spontaneous Raman scattering.

Visualisation of an nsPEF induced calcium wave using the genetically encoded calcium indicator GCaMP in U87 human glioblastoma cells.

Cytosolic, synthetic chemical calcium indicators are typically used to visualise the rapid increase in intracellular calcium ion concentration that follows nanosecond pulsed electric field (nsPEF) application. This study looks at the application of genetically encoded calcium indicators (GECIs) to investigate the spatiotemporal nature of nsPEF-induced calcium signals using fluorescent live cell imaging. Calcium responses to 44kV/cm, 10ns pulses were observed in U87-MG cells expressing either a plasma membrane targeted GECI (GCaMP5-G), or one cytosolically expressed (GCaMP6-S), and compared to the response of cells loaded with cytosolic or plasma membrane targeted chemical calcium indicators. Application of 100 pulses, to cells containing plasma membrane targeted indicators, revealed a wave of calcium across the cell initiating at the cathode side. A similar spatial wave was not observed with cytosolic indicators with mobile calcium buffering properties. The speed of the wave was related to pulse application frequency and it was not propagated by calcium induced calcium release.

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C.

The influence of infrared laser pulses on intracellular Ca2+ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca2+ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca2+ transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC50 of around 58 J.cm-2 ). For both type of cells, the source of the infrared-induced Ca2+ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP3 -induced Ca2+ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics.