A technique to detect and to quantify fasciocutaneous blood vessels in small laboratory animals ex vivo

PURPOSE: A microangiographical technique is described, which allows visualization of small and capillary blood vessels and quantification of fasciocutaneous blood vessels by means of digital computer analysis in very small laboratory animals. MATERIALS AND METHODS: The left carotid artery of 20 nu/nu mice was cannulated (26 gauge) and a mixture of gelatin, bariumsulfate, and green ink was injected according to standardized protocol. Fasciocutaneous blood vessels were visualized by digital mammography and analyzed for vessel length and vessel surface area as standardized units [SU] by computer program. RESULTS: With the described microangiography method, fasciocutaneous blood vessels down to capillary size level can be clearly visualized. Regions of interest (ROIs) can be defined and the containing vascular network quantified. Comparable results may be obtained by calculating the microvascular area index (MAI) and the microvascular length index (MLI), related to the ROIs size. Identical ROIs showed a high reproducibility for measured [SU] < 0.01 +/- 0.0012%. CONCLUSION: Combining microsurgical techniques, pharmacological knowledge, and modern digital image technology, we were able to visualize small and capillary blood vessels even in small laboratory animals. By using our own computer analytical program, quantification of vessels was reliable, highly reproducible, and fast.

Tolerance of human placental tissue to severe hypoxia and its relevance for dual ex vivo perfusion

In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20-30 min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30 min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage. There seem to be parallels between "depressed metabolism" seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by "partial metabolic arrest" in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress. There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin. A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the artificial reperfusion, may lead to an improvement of the ex vivo perfusion technique.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

BACKGROUND CONTEXT The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection. PURPOSE To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo. STUDY DESIGN In vitro and ex vivo whole-organ culture of hMSCs. METHODS In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype. RESULTS The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs. CONCLUSIONS Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.

Acute Stress Reduces Wound-Induced Activation of Microbicidal Potential of Ex Vivo Isolated Human Monocyte-Derived Macrophages

Background Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a) acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM), and (b) that these reductions are modulated by stress hormone release. Methods Fourty-one healthy men (mean age 35±13 years) were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker) before assessing HMDM microbicidal potential. Results Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p’s <.05). Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14–44.72). Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001). This effect was blocked by prior incubation with phentolamine. Conclusions Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

Development of an ex vivo protocol to model bone fracture in laying hens resulting from collisions.

Fractures of the keel bone, a bone extending ventrally from the sternum, are a serious health and welfare problem in free range laying hens. Recent findings suggest that a major cause of keel damage within extensive systems is collisions with internal housing structures, though investigative efforts have been hindered by difficulties in examining mechanisms and likely influencing factors at the moment of fracture. The objectives of this study were to develop an ex vivo impact protocol to model bone fracture in hens caused by collision, to assess impact and bird-related factors influencing fracture occurrence and severity, and to identify correlations of mechanical and structural properties between different skeletal sites. We induced keel bone fractures in euthanized hens using a drop-weight impact tester able to generate a range of impact energies, producing fractures that replicate those commonly found in commercial settings. The results demonstrated that impact energies of a similar order to those expected in normal housing were able to produce fractures, and that greater collision energies resulted in an increased likelihood of fractures and of greater severity. Relationships were also seen with keel's lateral surface bone mineral density, and the peak reactive force (strength) at the base of the manubrial spine. Correlations were also identified between the keel and long bones with respect to both strength and bone mineral density. This is the first study able to relate impact and bone characteristics with keel bone fracture at the moment of collision. Greater understanding of these relationships will provide means to reduce levels of breakage and severity in commercial systems.

Quantification of coronary artery stenosis with high-resolution CT in comparison with histopathology in an ex vivo study

PURPOSE To investigate the ex vivo performance of high-resolution computed tomography (CT) for quantitative assessment of percentage diameter stenosis in coronary arteries compared to histopathology. MATERIALS AND METHODS High-resolution CT was performed in 26 human heart specimens after the injection of iodinated contrast media into the coronary arteries. Coronary artery plaques were visually identified on CT images and the grade of stenosis for each plaque was measured with electronic calipers. All coronary plaques were characterized by histopathology according to the Stary classification, and the percentage of stenosis was measured. RESULTS CT depicted 84% (274/326) of all coronary plaques identified by histology. Missed plaques by CT were of Stary type I (n=31), type II (n=16), and type III (n=5). The stenosis degree significantly correlated between CT and histology (r=0.81, p<0.001). CT systematically overestimated the stenosis of calcified plaques (mean difference - 11.0 ± 9.5%, p<0.01) and systematically underestimated the stenosis of non-calcified plaques (mean difference -6.8 ± 10.4%, p<0.05), while there was no significant difference for mixed-type plaques (mean difference -0.4 ± 11.7%, p=0.85). There was a significant underestimation of stenosis degree as measured by CT for Stary II plaques (mean difference -14 ± 9%, p<0.01) and a significant overestimation for Stary VII plaques (mean difference 9 ± 10%, p<0.05), but there was no significant difference in stenosis degree between both modalities for other plaque types. CONCLUSIONS High-resolution CT reliably depicts advanced stage coronary plaques with an overall good correlation of stenosis degree compared to histology, however, the degree of stenosis is systematically overestimated in calcified and underestimated in non-calcified plaques.

Effects of ex vivo γ-tocopherol on airway macrophage function in healthy and mild allergic asthmatics

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate γ-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

Assessment of ultra-high resolution optical coherence tomography for monitoring tissue effects caused by laser photocoagulation of ex-vivo porcine retina

Retinal laser photocoagulation is an established and successful treatment for a variety of retinal diseases. While being a valuable treatment modality, laser photocoagulation shows the drawback of employing high energy lasers which are capable of physically destroying the neural retina. For reliable therapy, it is therefore crucial to closely monitor the therapy effects caused in the retinal tissue. A depth resolved representation of optical tissue properties as provided by optical coherence tomography may provide valuable information about the treatment effects in the retinal layers if recorded simultaneously to laser coagulation. Therefore, in this work, the use of ultra-high resolution optical coherence tomography to represent tissue changes caused by conventional and selective retinal photocoagulation is investigated. Laser lesions were placed on porcine retina ex-vivo using a 577 nm laser as well as a pulsed laser at 527 nm built for selective treatment of the retinal pigment epithelium. Applied energies were varied to generate lesions best representing the span from under- to overtreatment. The lesions were examined using a custom-designed optical coherence tomography system with an axial resolution of 1.78 μm and 70 kHz Ascan rate. Optical coherence tomography scans included volume scans before and after irradiation, as well as time lapse scans (Mscan) of the lesions. Results show OCT lesion visibility thresholds to be below the thresholds of ophthalmoscopic inspection. With the ultra-high resolution OCT, 42% - 44% of ophthalmoscopically invisible lesions could be detected and lesions that were under- or overexposed could be distinguished using the OCT data.

In vitro-ex vivo model systems for nanosafety assessment

Engineered nanomaterials have unique and novel properties enabling wide-ranging new applications in nearly all fields of research. As these new properties have raised concerns about potential adverse effects for the environment and human health, extensive efforts are underway to define reliable, cost- and time-effective, as well as mechanistic-based testing strategies to replace the current method of animal testing, which is still the most prevalent model used for the risk assessment of chemicals. Current approaches for nanomaterials follow this line. The aim of this review is to explore and qualify the relevance of new in vitro and ex vivo models in (nano)material safety assessment, a crucial prerequisite for translation into applications.

Syncytiotrophoblast vesicles show altered micro-RNA and haemoglobin content after ex-vivo perfusion of placentas with haemoglobin to mimic preeclampsia.

BACKGROUND Cell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content. METHODS The dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR. RESULTS All micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs. CONCLUSION This study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.

IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion.

BACKGROUND Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis. RESULTS Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/μL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/μL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin. CONCLUSIONS The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.