Confocal Blood Flow Videomicroscopy of Thrombus Formation over Human Arteries and Local Targeting of P2X7.

Atherothrombosis exposes vascular components to blood. Currently, new antithrombotic therapies are emerging. Herein we investigated thrombogenesis of human arteries with/without atherosclerosis, and the interaction of coagulation and vascular components, we and explored the anti-thrombogenic efficacy of blockade of the P2X purinoceptor 7 (P2X7). A confocal blood flow videomicroscopy system was performed on cryosections of internal mammary artery (IMA) or carotid plaque (CPL) determining/localizing platelets and fibrin. Blood from healthy donors elicited thrombi over arterial layers. Confocal microscopy associated thrombus with tissue presence of collagen type I, laminin, fibrin(ogen) and tissue factor (TF). The addition of antibodies blocking TF (aTF) or factor XI (aFXI) to blood significantly reduced fibrin deposition, variable platelet aggregation and aTF + aFXI almost abolished thrombus formation, showing synergy between coagulation pathways. A scarce effect of aTF over sub-endothelial regions, more abundant in tissue TF and bundles of laminin and collagen type I than deep intima, may suggest tissue thrombogenicity as molecular structure-related. Consistently with TF-related vascular function and expression of P2X7, the sections from CPL but not IMA tissue cultures pre-treated with the P2X7 antagonist A740003 demonstrated poor thrombogenesis in flow experiments. These data hint to local targeting studies on P2X7 modulation for atherothrombosis prevention/therapy.

Evaluation of Endothelial Dysfunction and Autophagy in Fibromyalgia-Related Vascular and Cerebral Cortical Changes and the Ameliorative Effect of Fisetin.

Fibromyalgia (FM) is a common chronic pain syndrome that affects 1% to 5% of the population. We aimed to investigate the role of endothelial dysfunction and autophagy in fibromyalgia-related vascular and cerebral cortical changes in a reserpine-induced rat model of fibromyalgia at the histological and molecular levels and to study the ameliorative effect of fisetin. Forty adult female albino rats were divided into four groups (10 each): two control groups, the reserpine-induced fibromyalgia group, and the fisetin-treated group. The carotid arteries and brains of the animals were dissected. Frozen tissue samples were used for total RNA extraction and qPCR analysis of eNOS, caspase-3, Bcl-2, LC-3, BECN-1, CHOP, and TNF-α expression. Histological, immunohistochemical (eNOS), and ultrastructure studies were conducted. The carotid arteries revealed excessive autophagy and endothelial, vascular, and apoptotic changes. The cerebral cortex showed similar findings apart from endoplasmic reticulum stress. Additionally, there was decreased gene expression of eNOS and Bcl-2 and increased expression of caspase-3, LC-3, BECN-1, CHOP, and TNF-α. In the fisetin-treated rats, improvements in the histological and molecular results were detected. In conclusion, oxidative stress, enhanced apoptosis, and excessive autophagy are fundamental pathophysiologic mechanisms of reserpine-induced fibromyalgia. Moreover, fisetin has an ameliorative effect against fibromyalgia.

Enhancing medial layer recellularization of tissue-engineered blood vessels using radial microchannels.

Aim: Cell repopulation of tissue-engineered vascular grafts (TEVGs) from decellularized arterial scaffolds is limited by dense concentric tunica media layers which impede cells migrating radially between the layers. We aimed to develop and validate a new microneedle device to modify decellularized carotid arteries with radial microchannels to enhance medial layer repopulation. Material & methods: Modified decellularized porcine arteries were seeded with rat mesenchymal stem cells using either standard longitudinal injection, or a dual vacuum-perfusion bioreactor. Mechanical tests were used to assess the arterial integrity following modification. Results & conclusion: The method herein achieved radial recellularization of arteries in vitro without significant loss of mechanical integrity, Thus, we report a novel method for successful radial repopulation of decellularized carotid artery-based tissue-engineered vascular grafts.

Decellularization, cross-linking and heparin immobilization of porcine carotid arteries for tissue engineering vascular grafts.

Tissue engineering vascular grafts (TEVGs) have the potential to replace small-diameter grafts in bypass surgery which is good news for patients with cardiovascular disease. Decellularized arteries can be ideal TEVGs because their natural three-dimensional structures support the migration of host cells and vascular remodeling. There are many methods for decellularization without a standard protocol. In this study, a combination of Triton X-100 and sodium dodecyl sulfate (SDS) were used to prepare decellularized arteries. However, decellularization may damage the biochemical and mechanical properties to some degree. We used the cross-linking agents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to improve mechanical properties and immobilize heparin to inhibit thrombogenesis. Histological analysis, scanning electron microscopy, biomechanical properties test, determination of immobilized heparin, active partial thrombin time assay, and subcutaneous embedding experiment were used to evaluate the efficiency of decellularization and the efficacy of heparinized cross-linked vascular scaffold. Results showed 1% Triton X-100 combined with 0.3% SDS can decellularize successfully. EDC and NHS cross-linking can improve the mechanical properties, reduce the inflammatory reaction and slow the degradation time. Heparin immobilized on the scaffolds can inhibit thrombogenesis effectively. This study indicated the heparinized cross-linked vascular scaffolds may be ideal scaffolds for TEVGs.

Combination of freeze-thaw with detergents: A promising approach to the decellularization of porcine carotid arteries.

BACKGROUND: A tissue engineering technique based on use of the extracellular matrix (ECM) as a scaffold shows great potential for preparing small-caliber vascular grafts. Decellularization protocols are still not standardized for bioengineering. The effects of freeze-thaw cycles used for decellularization are unknown. OBJECTIVE: To evaluate the effects of freeze-thaw cycles on porcine carotid arteries during decellularization and to develop a promising protocol for preparing ECM scaffolds. METHODS: Porcine carotid arteries were decellularized with freeze-thaw cycles followed by three different chemical protocols. Histological analysis, scanning electron microscopy, mechanical tests and pore size measurement were performed to assess their effects on the ECM. RESULTS: The composition, structure, and mechanical properties were not significantly changed after freeze-thaw cycles, with the exception of endothelial cells loss. Freeze-thaw led to a porous structure within arteries. The use of Triton X-100 followed by sodium dodecyl sulfate (SDS) resulted in ECM scaffolds with well-preserved composition, structure, and mechanical properties, as well as with adequate porosity. CONCLUSIONS: As the initial step for decellularization, freeze-thaw had little impact on arteries. Decellularized porcine carotid arteries, prepared using freeze-thaw cycles followed by treatment with Triton X-100 and SDS, may serve as a promising biological scaffold as a tissue-engineered vascular graft.

Development of long in vivo tissue-engineered "Biotube" vascular grafts.

In-body tissue architecture (iBTA), a cell-free, in vivo tissue engineering technology that can produce autologous implantable tissues of the desired shape by subcutaneously embedding specially designed molds, was used to develop long tubular collagenous tissues called Biotubes. Spiral molds for long Biotubes were assembled with an outer pipe-shaped spiral shell and an inner spiral mandrel, and embedded into subcutaneous pouches of beagle dogs or goats for 1 or 2 months. Tubular collagenous tissues were formed at the space between the shell and the mandrel of the mold. Depending on the spiral turn number in the mold, Biotubes of 25 cm or 50 cm (internal diameter 4 mm or 5 mm) were prepared with nearly homogeneous mechanical and histological properties over their entire length. Biotubes stored in 70% ethanol were allogenically implanted into beagle dogs or goats to evaluate their in vivo performance. The 25-cm Biotubes functioned as arterial grafts with no need for luminal modification or mechanical support, and demonstrated vascular reconstruction within 3 months after implantation into dogs. The 50-cm Biotubes functioned as arteriovenous shunt grafts in the neck region of goats without thrombus formation and vascular deformation for 1 month. Thus, the world's longest tissue-engineered vascular grafts with small diameter could be developed using iBTA.

Preparation and evaluation of decellularized porcine carotid arteries cross-linked by genipin: the preliminary results.

Decellularized arteries have been considered as promising scaffolds for small-diameter vascular substitutes. However, weakened mechanical properties, immunological rejection and rapid degradation after transplantation still exist after decellularization. Previous studies indicated that genipin cross-linking can solve these problems. Therefore, genipin was selected as the cross-linking agent for the pre-treatment of decellularized arteries in our study. Histological analysis, scanning electron microscopy, mechanical properties analysis and subcutaneous embedding experiment were adopted to investigate the efficiency of decellularization and the effect of genipin cross-linking on improving mechanical, structural and biological properties of decellularized arteries. Decellularization protocols based on three trypsin concentrations were used to prepare decellularized arteries, after decellularization, arteries were cross-linked with genipin. Results showed that 0.5% trypsin was the most efficient concentration to remove cellular components and preserve ECM. However, mechanical properties of 0.5% trypsin decellularized arteries weakened significantly, while genipin cross-linking improved mechanical properties of decellularized arteries to the same level as fresh arteries. After 4 weeks subcutaneous embedding, cross-linked arteries caused the mildest inflammatory response. In conclusion, genipin could be employed as an ideal cross-linking agent to strengthen mechanical properties, enhance the resistance to degradation and reduce the antigenicity of decellularized arteries for small-diameter blood vessel tissue engineering applications.

Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

Quantifying the ultrastructure of carotid arteries using high-resolution micro-diffusion tensor imaging-comparison of intact versus open cut tissue.

Diffusion magnetic resonance imaging (dMRI) can provide insights into the microstructure of intact arterial tissue. The current study employed high magnetic field MRI to obtain ultra-high resolution dMRI at an isotropic voxel resolution of 117 µm3 in less than 2 h of scan time. A parameter selective single shell (128 directions) diffusion-encoding scheme based on Stejskel-Tanner sequence with echo-planar imaging (EPI) readout was used. EPI segmentation was used to reduce the echo time (TE) and to minimise the susceptibility-induced artefacts. The study utilised the dMRI analysis with diffusion tensor imaging (DTI) framework to investigate structural heterogeneity in intact arterial tissue and to quantify variations in tissue composition when the tissue is cut open and flattened. For intact arterial samples, the region of interest base comparison showed significant differences in fractional anisotropy and mean diffusivity across the media layer (p < 0.05). For open cut flat samples, DTI based directionally invariant indices did not show significant differences across the media layer. For intact samples, fibre tractography based indices such as calculated helical angle and fibre dispersion showed near circumferential alignment and a high degree of fibre dispersion, respectively. This study demonstrates the feasibility of fast dMRI acquisition with ultra-high spatial and angular resolution at 7 T. Using the optimised sequence parameters, this study shows that DTI based markers are sensitive to local structural changes in intact arterial tissue samples and these markers may have clinical relevance in the diagnosis of atherosclerosis and aneurysm.

Cerebral Venous Collagen Remodeling in a Modified White Matter Lesions Animal Model.

To mimic the expected pathological changes of white matter lesions (WMLs) and increase the stability, we applied modified two-vessel occlusion (modified 2VO) (1-week interval bilateral carotid artery occlusion) in stroke-prone renovascular hypertensive rats (RHRSP) and established a modified WMLs model (RHRSP/modified 2VO) that compared their phenotypes with RHRSP and sham-operated rats. In addition, we tried to differentiate small veins from small arteries through the presence of smooth muscle to study the pathological changes of small veins detailed in the model. RHRSP/modified 2VO rats showed higher stability and more extensive white matter damage without an obvious increase in mortality rate at 12 weeks after the modified 2VO operation compared to RHRSP rats. RHRSP/modified 2VO rats showed more severe small venous collagen deposition than RHRSP rats, and the majority of the deposition was collagen I and IV rather than collagen III. In addition, RHRSP/modified 2VO rats possessed cognitive impairment, mild wall thickness and blood-brain barrier disruption. Our findings suggest that the modified WMLs model (RHRSP/modified 2VO) mimics cognitive impairment and small vessel pathological changes similar to WMLs in humans. Differentiating small veins from small arteries through smooth muscle is feasible, and marked small venous deposition may play an important role in the hypertensive white matter lesions.

Morphologic mechanisms of increased vascular permeability of triolein emulsion to the blood-brain barrier.

Triolein emulsion has been known to increase vascular permeability in the brain when it is infused into the carotid artery. The purpose of this study was to identify the morphologic mechanism of increased vascular permeability in brain induced by infusion of emulsified triolein into the carotid artery by transmission electron microscopy (TEM). Triolein emulsion was infused into the carotid artery of rats. TEM using lanthanum tracer was used to evaluate morphologic changes in endothelium with a focus on transcytotic vesicles and tight junction opening. The treat group showed multiple transcytotic vesicles containing lanthanum tracer within endothelium on TEM. TEM also revealed that lanthanum tracer entered neural interstitium through tight junctions between capillary endothelial cells infrequently in the treat group. No evidence of transcytotic vesicles containing lanthanum tracer or lanthanum leakage through tight junctions was observed in the control group. Transcytosis and the opening of tight junctions appears the pathway for vascular permeability enhancement by triolein. This result could be utilized in studies on the blood-brain barrier and by those searching for chemotherapeutic methods that deliver anti-tumor agents to normally drug inaccessible organs.

Carotid plaque instability is not related to quantity but to elemental composition of calcification.

BACKGROUND AND AIMS: Recent studies highlighted the role of calcification processes in the clinical progression of chronic cardiovascular diseases. In this study we investigated the relationship between the chemical composition of calcification and atherosclerotic plaque stability in carotid arteries. METHODS AND RESULTS: To this end, we characterized the calcification on 229 carotid plaques, by morphology, immunohistochemistry, transmission electron microscopy and energy dispersive X-ray microanalysis. Plaques were classified into two categories: unstable and stable. No significant differences were found in the incidence of the various risk factors between patients with and without carotid calcification, with the exception of diabetes. The energy dispersive X-ray microanalysis allowed us to identify two types of calcium salts in the atheromatous plaques, hydroxyapatite (HA) and calcium oxalate (CO). Our results showed that calcification is a common finding in carotid plaques, being present in 77.3% of cases, and the amount of calcium is not a factor of vulnerability. Noteworthy, we observed an association between HA calcification and unstable plaques. On the contrary, CO calcifications were mainly detected in stable plaques. CONCLUSIONS: The presence of different types of calcification in atheromatous plaques may open new perspectives in understanding the molecular mechanisms of atheroma formation and plaque instability.

Poly(ethylmethacrylate-co-diethylaminoethyl acrylate) coating improves endothelial re-population, bio-mechanical and anti-thrombogenic properties of decellularized carotid arteries for blood vessel replacement.

Decellularized vascular scaffolds are promising materials for vessel replacements. However, despite the natural origin of decellularized vessels, issues such as biomechanical incompatibility, immunogenicity risks and the hazards of thrombus formation, still need to be addressed. In this study, we coated decellularized vessels obtained from porcine carotid arteries with poly (ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) with the purpose of improving endothelial coverage and minimizing platelet attachment while enhancing the mechanical properties of the decellularized vascular scaffolds. The polymer facilitated binding of endothelial cells (ECs) with high affinity and also induced endothelial cell capillary tube formation. In addition, platelets showed reduced adhesion on the polymer under flow conditions. Moreover, the coating of the decellularized arteries improved biomechanical properties by increasing its tensile strength and load. In addition, after 5 days in culture, ECs seeded on the luminal surface of 8g7-coated decellularized arteries showed good regeneration of the endothelium. Overall, this study shows that polymer coating of decellularized vessels provides a new strategy to improve re-endothelialization of vascular grafts, maintaining or enhancing mechanical properties while reducing the risk of thrombogenesis. These results could have potential applications in improving tissue-engineered vascular grafts for cardiovascular therapies with small caliber vessels.

Histopathologic Validation of Grayscale Carotid Plaque Characteristics Related to Plaque Vulnerability.

Inflammation and angiogenesis play major roles in carotid plaque vulnerability. The purpose of this study was to determine whether gray-scale features of carotid plaques are associated with histologic markers for inflammation. Thirty-eight individuals completed a dedicated research carotid ultrasound exam before carotid endarterectomy. Gray-scale analysis was performed on plaque images to measure plaque echogenicity (gray-scale median [GSM] pixel brightness), plaque area, presence of discrete white areas (DWAs) and the percent of black area near the lumen on any one component of the plaque. Plaques with higher ultrasound GSM had greater percent calcification (p = 0.013) on histopathology. Presence of an ultrasound DWA was associated with more plaque hemosiderin (p = 0.0005) and inflammation (p = 0.019) on histopathology examination. The percent of plaque black area in any one component was associated with a higher score for macroscopic ulceration (p = 0.028). Ultrasound plaque characteristics (GSM, DWAs and black areas) represent histopathologic markers associated with plaque vulnerability. ClinicalTrials.gov identifier: NCT02476396.

Towards the characterisation of carotid plaque tissue toughness: Linking mechanical properties to plaque composition.

UNLABELLED: The morphological manifestation of calcification within an atherosclerotic plaque is diverse and the response to cutting balloon angioplasty remains an elusive target to predict in the presence of extensive calcification. This study examines the resistance of plaque tissue to blade penetration by characterising the underlying toughness properties and stratifying the upper and lower scale toughness limits based on the strong mechanical influence of calcification. Mechanical toughness properties of the common, bifurcation and internal carotid artery (n=62) were determined using guillotine-cutting tests measuring the energy required to pass a surgical blade through a unit length of plaque tissue. The corresponding structural composition of the dissected plaque segments was characterised using Fourier transform infrared analysis, electron microscopy and energy dispersive x-ray spectroscopy. Mechanical results reveal a clear distinction in toughness properties within each region of the carotid vessel with significantly tougher properties localised in the bifurcation (p=0.004) and internal region (p=0.0003) compared to the common. The severity of the intra-plaque variance is highest in plaques with high toughness localised in the bifurcation region (p<0.05). Structural examination reveals that the diverse mechanical influence of the level of calcification present is characteristic of specific regions within the carotid plaque. The energy required to overcome the calcific resistance and propagate a controlled cut in the calcified tissue at each region varies further with the degree of plaque progression. The identification of the localised calcification characteristics is a key determinant in achieving successful dissection of the severely toughened plaque segments during cutting balloon angioplasty. STATEMENT OF SIGNIFICANCE: Calcification plays a fundamental role in plaque tissue mechanics and demonstrates a diverse range of material moduli properties. This work addresses the characterisation of the toughness properties in human carotid plaque tissue using a fracture mechanics approach. Toughness determines the energy required to propagate a controlled cut in the plaque material. This parameter is crucial for predicting the cutting forces required during endovascular cutting balloon angioplasty intervention. Results demonstrate that a strong relationship exists between the structural calcification configurations, fracture mechanisms and associated toughness properties that are characteristic of specific regions within the carotid artery plaque. The identification of the morphological characteristics of localised calcification may serve as a valuable quantitative measure for cutting balloon angioplasty treatment.

Real-Time Elastography Visualization and Histopathological Characterization of Rabbit Atherosclerotic Carotid Arteries.

To evaluate the feasibility of non-invasive vascular real-time elastography imaging (RTE) in visualizing the composition of rabbit carotid atherosclerotic plaque as determined by histopathology, a rabbit model of accelerated carotid atherosclerosis was used. Thirty rabbits were randomly divided into two groups of 15 rabbits each. The first group was fed a cholesterol-rich diet and received balloon-induced injury the left common carotid artery endothelium, whereas the second group only received a cholesterol-rich diet. The rabbits were all examined in vivo with HITACHI non-invasive vascular real-time elastography (Hi-RTE) at baseline and 12 wk, and results from the elastography were compared with American Heart Association histologic classifications. Hi-RTE and the American Heart Association histologic classifications had good agreement, with weighted Cohen's kappa (95% confidence internal) of 0.785 (0.649-0.920). Strains of segmented plaques that were stained in different colors were statistically different (p < 0.0001). The sensitivity and specificity of elastograms for detecting a lipid core were 95.5% and 61.5%, respectively, and the area under the receiver operating characteristic curve was 0.789, with a 95% confidence interval of 0.679 to 0.876. This study is the first to indicate the feasibility of utilizing Hi-RTE in visualizing normal and atherosclerotic rabbit carotid arteries non-invasively. This affordable and reliable method can be widely applied in research of both animal and human peripheral artery atherosclerosis.

Carotid artery distensibility and hormone therapy and menopause: the Los Angeles Atherosclerosis Study.

OBJECTIVE: Observational studies have suggested that arterial distensibility decreases during menopause; however, its relationship with hormone therapy use remains controversial. We prospectively studied distensibility and hormone therapy use at different menopause stages. METHODS: One hundred sixty-one women (aged between 42 and 61 y) without cardiovascular disease underwent carotid artery measurements by ultrasound to calculate distensibility index at baseline and 3 years later. Menopause stage was classified at each visit as premenopausal, perimenopausal, and postmenopausal. Across 3 years of prospective observation, women were classified as remaining premenopausal, remaining postmenopausal, or transitioning (defined as change from premenopausal to perimenopausal, from premenopausal to postmenopausal, from perimenopausal to perimenopausal, or from perimenopausal to postmenopausal). RESULTS: Distensibility declined across time at all menopause stages (P < 0.0001). Compared with postmenopausal women, premenopausal and transitioning/no hormone therapy women had more than twice the decline in distensibility index (P = 0.06 and P = 0.016, respectively), whereas transitioning/hormone therapy women did not differ in distensibility decline (P = 0.28). In a multivariate model, change in systolic blood pressure (P < 0.0001) and change in pulse pressure (P = 0.004) were independent predictors of distensibility index change and served as effect modulators. In an adjusted model, women in the premenopausal and transitioning/no hormone therapy groups had a significantly faster decline in distensibility index (P = 0.002 and P = 0.001, respectively) compared with postmenopausal women, whereas the transitioning/hormone therapy group did not (P = 0.21). CONCLUSIONS: These findings confirm that the menopausal transition is associated with reduced vascular compliance. Hormone therapy is associated with better arterial distensibility only during the menopausal transition. Additional prospective studies are needed to confirm these findings and to determine whether hormone therapy use beyond the menopausal transition is related to distensibility.

Kinetics of endothelialization of the multilayer flow modulator and single-layer arterial stents.

The multilayer flow modulator (MFM; Cardiatis, Isnes, Belgium) is a self-expandable mesh of braided cobalt alloy wires, used for treatment of aortic and peripheral aneurysms. To further improve our understanding of this novel technology, the endothelialization kinetics of the MFM was investigated and compared with those of two marketed single-layer stents. Five porcine animal models were used in which a total of 19 stents were implanted in the iliac and carotid arteries between one and five weeks before sacrifice. All 19 stents were successfully delivered. For all devices, nonsignificant signs of inflammation or thrombosis were noted, and there was no evidence of local intolerance. The MFM developed a thin layer of endothelial cells earlier and was associated with less neointimal development than the two single-layer stents. A differing phenomenon of integration was also revealed and hypothesized as endothelialization from adhesion of circulating endothelial progenitor cells, as well as adhesion from the arterial wall, and also by the differences in trauma exposed to the arterial wall.

A Novel Vascular Coupling System for End-to-End Anastomosis.

Vascular anastomosis is common during reconstructive surgeries. Traditional hand-suturing techniques are time consuming, subject to human error, and require high technical expertise and complex instruments. Prior attempts to replace hand-suturing technique, including staples, ring-pin devices, cuffing devices, and clips, are either more cumbersome, are unable to maintain a tight seal, or do not work for both arteries and veins. To provide a more efficient and reliable vessel anastomosis, a metal-free vascular coupling system that can be used for both arteries and veins was designed, fabricated and tested. A set of corresponding instruments were developed to facilitate the anastomosis process. Evaluation of the anastomosis by scanning electron microscopy and magnetic resonance imaging, demonstrated that the installation process does not cause damage to the vessel intima and the vascular coupling system is not exposed to the vessel lumen. Mechanical testing results showed that vessels reconnected with the vascular coupling system could withstand 12.7 ± 2.2 N tensile force and have superior leak profiles (0.049 ± 0.015, 0.078 ± 0.016, 0.089 ± 0.008 mL/s at 160, 260, 360 mmHg, respectively) compared to hand sutured vessels (0.310 ± 0.014, 1.123 ± 0.033, 2.092 ± 0.072 mL/s at 160, 260, 360 mmHg, respectively). The anastomotic process was successfully demonstrated on both arteries and veins in cadaver pigs.

Electron microscopic aspects of the effects of certain prostaglandin analogs on mouse testes.

Prostaglandins were highlighted in the seminal plasma and then in the rest of the male and female genital tract. Prostaglandin analogs, firstly used in obstetrics and gynecology, are now widespread in both sexes, especially in the treatment of gastric and duodenal ulcers, glaucoma, etc. Therefore, we tried to highlight the effects of repeated administration of Cloprostenol and CIPG isopropyl ester (both prostaglandin F2α analogs) for the male gonad. In our experiment, we used Cloprostenol and CIPG isopropyl ester. We used three groups of white, male mice, aged 50-80 days, kept in standard laboratory conditions, which received the same feed. Each group included 12 mice. The first batch was the control group and received no substance at all. The second batch received 25 µg/kg of Cloprostenol dose per body per day, intraperitoneal administration (a single dose per day) on a daily basis for a four weeks period of time. The third batch received a 25 µg/kg CIPG isopropyl ester dose per body/day intraperitoneal administration (a single dose per day) on a daily basis for a four weeks period of time. After 7, 14 and 28 days of treatment, we sacrificed four animals in each of the batches by cutting their carotid arteries. The prostanoid analogs we used, Cloprostenol and CIPG isopropyl ester, have similar actions on male gonad in mice. These analogs induced significant changes in the evolution of the spermatogenesis and spermiogenesis. In relation to the treatment duration there were cellular changes suggesting apoptosis in different stages. With regard to spermiogenesis, the ultrastructural aspects indicate a decrease of the sperm structuring processes, especially in the acrosomal apparatus and chromatin.