Treatment of Pythiosis in Equine Limbs Using Intravenous Regional Perfusion of Amphotericin B

Objective To evaluate the effects of intravenous regional limb perfusion (IRLP) administration of amphotericin B in horses to treat pythiosis after surgical excision and thermocautery. Study Design Case series. Animals Horses (n = 12) with Pythium insidiosum infection of the distal aspect of the thoracic or pelvic limbs. Methods After surgical excision of granulation tissue and thermocautery, 50 mg amphotericin B was administered by IRLP through a catheter placed in a superficial vein of the affected limb next to the lesion after placing a tourniquet above the injection site. The lesions and locomotor system were evaluated before treatment and at 7, 14, 21, 28, 35, and 60 days. Results Ninety-two percent of horses treated with amphotericin B had complete lesion resolution 35 or 60 days after 1 or 2 IRLP treatments, respectively. IRLP induced limb edema and pain during regional palpation in 42%, and inflammation of the injection site in 33% of horses; however these signs resolved after 14 days. Conclusions IRLP administration of amphotericin B was effective for treating pythiosis in equine limbs, resolving infection with manageable side effects.

Influence of different routes of flush perfusion on the distribution of lung preservation solutions in parenchyma and airways

Objective: The present study was performed to investigate the influence of different routes of perfusion on the distribution of the preservation solutions in the lung parenchyma and upper airways. Methods: Pigs were divided into four groups: control (n = 6), pulmonary artery (PA) (n = 6), simultaneous PA + bronchial artery (BA) (n = 8), and retrograde delivery (n = 6). After preparation and cannulation, cardioplegia solution and Euro- Collins solution (ECS) for lung preservation were given simultaneously. After removal of the heart, the double lung bloc was harvested. Following parameters were assessed: total and regional perfusion (dye-labeled microspheres), tissue water content, PA, aorta, left atrial and left ventricular pressures, cardiac output and lung temperature. Results: Our data show that flow of the ECS in lung parenchyma did not reach control values (9.4 ± 1.0 ml/min per g lung wet weight) regardless of the route of delivery (PA 6.3 ± 1.5, PA + BA 4.8 ± 0.9, retrograde 2.7 ± 0.9 ml/min per g lung wet weight). However, flow in the proximal and distal trachea were significantly increased by PA + BA delivery (0.970 ± 0.4, respectively, 0.380 ± 0.2 ml/min per g) in comparison with PA (0.023 ± 0.007, respectively, 0.024 ± 0.070 ml/min per g), retrograde (0.009 ± 0.003, respectively, 0.021 ± 0.006 ml/min per g) and control experiments (0.125 ± 0.0018, respectively, 0.105 ± 0.012 ml/g per min). Similarly the highest flow rates in the right main bronchus were achieved by PA + BA delivery (1.04 ± 0.4 ml/min per g) in comparison with 0.11 ± 0.03 in control, 0.033 ± 0.008 in PA, and 0.019 ± 0.005 ml/min per g in retrograde group. Flows in the left main bronchus were 0.09 ± 0.02 ml/min per g in control, 0.045 ± 0.012 ml/min per g in PA, and 0.027 ± 0.006 ml/min per g in retrograde group. The flow rates were significantly (P = 0.001) increased by PA + BA delivery of the storage solution (0.97 ± 0.3 ml/min per g). Conclusions: Our data show that the distribution of ECS for lung preservation is significantly improved in airway tissues (trachea and bronchi) if a simultaneous PA + BA delivery is used.

Histologic and functional evaluation of lungs reconditioned by ex vivo lung perfusion

BACKGROUND: Only about 15% of donor lungs are considered suitable for transplantation (LTx). Ex vivo lung perfusion (EVLP) has been developed as a method to reassess and repair damaged lungs. We report our experience with EVLP in non-acceptable donor lungs and evaluate its ability to recondition these lungs. METHODS: We studied lungs from 16 brain-dead donors rejected for LTx. After harvesting, the lungs were stored at 4 degrees C for 10 hours and subjected to normothermic EVLP with Steen Solution (Vitro life, Goteborg, Sweden) for 60 minutes. For functional evaluation, the following variables were assessed: partial pressure of arterial oxygen (Pao(2)), pulmonary vascular resistance (PVR), and lung compliance (LC). For histologic assessment, lung biopsy was done before harvest and after EVLP. Tissue samples were examined under light microscopy. To detect and quantify apoptosis, terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling assay was used. RESULTS: Thirteen lima donors were refused for having impaired lung function. The mean Pao(2) obtained in the organ donor at the referring hospital was 193.7 mm Hg and rose to 489 mm Hg after EVLP. During EVLP, the mean PVR was 652.5 dynes/sec/cm(5) and the mean LC was 48 ml/cm H2O. There was no significant difference between the mean Lung Injury Score before harvest and after EVLP. There was a trend toward a reduction in the median number of apoptotic cells after EVLP. CONCLUSIONS: EVLP improved lung function (oxygenation capacity) of organs considered unsuitable for transplantation. Lung tissue structure did not deteriorate even after 1 hour of normothermic perfusion. J Heart Lung Transplant 2012;31:305-9 (C) 2012 International Society for Heart and Lung Transplantation. All rights reserved.

Treatment of Pythiosis in Equine Limbs Using Intravenous Regional Perfusion of Amphotericin B

Objective To evaluate the effects of intravenous regional limb perfusion (IRLP) administration of amphotericin B in horses to treat pythiosis after surgical excision and thermocautery. Study Design Case series. Animals Horses (n = 12) with Pythium insidiosum infection of the distal aspect of the thoracic or pelvic limbs. Methods After surgical excision of granulation tissue and thermocautery, 50 mg amphotericin B was administered by IRLP through a catheter placed in a superficial vein of the affected limb next to the lesion after placing a tourniquet above the injection site. The lesions and locomotor system were evaluated before treatment and at 7, 14, 21, 28, 35, and 60 days. Results Ninety-two percent of horses treated with amphotericin B had complete lesion resolution 35 or 60 days after 1 or 2 IRLP treatments, respectively. IRLP induced limb edema and pain during regional palpation in 42%, and inflammation of the injection site in 33% of horses; however these signs resolved after 14 days. Conclusions IRLP administration of amphotericin B was effective for treating pythiosis in equine limbs, resolving infection with manageable side effects.

Finite element modeling of flow/compression-induced deformation of alginate scaffolds for bone tissue engineering

Trauma or degenerative diseases such as osteonecrosis may determine bone loss whose recover is promised by a "tissue engineering“ approach. This strategy involves the use of stem cells, grown onboard of adequate biocompatible/bioreabsorbable hosting templates (usually defined as scaffolds) and cultured in specific dynamic environments afforded by differentiation-inducing actuators (usually defined as bioreactors) to produce implantable tissue constructs. The purpose of this thesis is to evaluate, by finite element modeling of flow/compression-induced deformation, alginate scaffolds intended for bone tissue engineering. This work was conducted at the Biomechanics Laboratory of the Institute of Biomedical and Neural Engineering of the Reykjavik University of Iceland. In this respect, Comsol Multiphysics 5.1 simulations were carried out to approximate the loads over alginate 3D matrices under perfusion, compression and perfusion+compression, when varyingalginate pore size and flow/compression regimen. The results of the simulations show that the shear forces in the matrix of the scaffold increase coherently with the increase in flow and load, and decrease with the increase of the pore size. Flow and load rates suggested for proper osteogenic cell differentiation are reported.

Preservation of Amputated Extremities by Extracorporeal Blood Perfusion; a Feasibility Study in a Porcine Model

Successful extremity transplantations and replantations have to be performed within 6 h of amputation to avoid irreversible tissue loss. This study investigates ex vivo the technical feasibility and the limb preservation potential of extracorporeal whole blood perfusion in a porcine model.

Rapidly excised and cryofixed rat tissue

All preparation efforts of biological samples in electron microscopy are focused to preserve structures as close as possible to the native state. To achieve this goal with tissues, it is of advantage to have a very short time between excision and fixation. The most common approach is chemical fixation: cross-linking of the tissue samples with aldehydes followed by postfixation with osmium tetroxide. Here, the fastest approach for tissue samples is perfusion. However, the diffusion of the fixation solution from blood vessels into the depth of the tissue is still slow and does not allow an overall instant fixation of a single cell. As a result, osmotic effects become evident (swelling or shrinkage of cell organelles). Another possibility is to take a tissue sample from the experimental animal. Excision of tissue can last quite some time, which results in even more pronounced autolytic induced osmotic effects. Furthermore, the animal does not survive the procedure in most cases. Alternatively, microbiopsies are an elegant technique to rapidly excise small quantities of tissue. Some tissues, such as liver and muscle, may be obtained using a non-lethal approach. To avoid the artifacts introduced by chemical fixation, high-pressure freezing of microbiopsies (brain, liver, kidney, and muscle) is a powerful alternative to chemical fixation. Here, we describe the microbiopsy method, and high-pressure freezing/freeze-substitution (HPF/FS) as a follow-up procedure. Cryosectioning of high-pressure frozen samples is optimally preserving the ultrastructure; however, it is not considered to be a routine approach yet.

Histological evidence of delayed ischemic brain tissue damage in the rat double-hemorrhage model

The rat double-SAH model is one of the standard models to simulate delayed cerebral vasospasm (CVS) in humans. However, the proof of delayed ischemic brain damage is missing so far. Our objective was, therefore, to determine histological changes in correlation with the development of symptomatic and perfusion weighted imaging (PWI) proven CVS in this animal model. CVS was induced by injection of autologous blood in the cisterna magna of 22 Sprague-Dawley rats. Histological changes were analyzed on day 3 and day 5. Cerebral blood flow (CBF) was assessed by PWI at 3 tesla magnetic resonance (MR) tomography. Neuronal cell count did not differ between sham operated and SAH rats in the hippocampus and the cerebral cortex on day 3. In contrast, on day 5 after SAH the neuronal cell count was significantly reduced in the hippocampus (p<0.001) and the inner cortical layer (p=0.03). The present investigation provides quantitative data on brain tissue damage in association with delayed CVS for the first time in a rat SAH model. Accordingly, our data suggest that the rat double-SAH model may be suitable to mimic delayed ischemic brain damage due to CVS and to investigate the neuroprotective effects of drugs.

High-quality lung fixation by controlled closed loop perfusion for stereological analysis in a large animal model

Stereology is an essential method for quantitative analysis of lung structure. Adequate fixation is a prerequisite for stereological analysis to avoid bias in pulmonary tissue, dimensions and structural details. We present a technique for in situ fixation of large animal lungs for stereological analysis, based on closed loop perfusion fixation.

C-reactive protein production in term human placental tissue

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Differential release of plasminogen activator inhibitors (PAIs) during dual perfusion of human placenta: implications in preeclampsia

Plasminogen activator inhibitors (PAIs) play critical roles in regulating cellular invasion and fibrinolysis. An increase in the ratio of PAI-1/PAI-2 in placenta and maternal serum is suggested to result in excessive intervillous fibrin deposition and placental infarction in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). In the current study we used dual (maternal and fetal) perfusion of human term placentas to examine the release of PAIs to the intervillous space. ELISA revealed a significant time-dependent increase in total PAI-1 levels in maternal perfusate (MP) between 1 and 7h of perfusion. Conversely, PAI-2 levels decreased resulting in a 3-fold increase in the PAI-1/PAI-2 ratio in MP. Levels of PAI-1, but not PAI-2, in placental tissue extracts increased during perfusion. In perfusions carried out with xanthine and xanthine oxidase (X + XO), compounds used to generate reactive oxygen species (ROS), no time-dependent increase in total PAI-1 levels was observed. In addition, X + XO treatment promoted a 3-fold reduction in active PAI-1 levels in MP, indicating that ROS decrease PAI-1 release to MP. The finding of a time-dependent change in patterns of PAI expression and response to ROS indicates the utility of dual perfusion as a model to dissect mechanism(s) promoting aberrant fibrinolysis in pregnancies complicated by PE and IUGR.

Effects of continuous remifentanil administration on intra-operative subcutaneous tissue oxygen tension

Surgical stress response markedly increases sympathetic nerve activity and catecholamine concentrations. This may contribute to peripheral vasoconstriction, reduced wound perfusion and subsequent tissue hypoxia. Opioids are known to depress the hypothalamic-adrenal response to surgery in a dose-dependent manner. We tested the hypothesis that continuous remifentanil administration produces improved subcutaneous tissue oxygen tension compared to fentanyl bolus administration. Forty-six patients undergoing major abdominal surgery were randomly assigned to receive either fentanyl bolus administration or continuous remifentanil infusion. Mean subcutaneous tissue oxygen values over the entire intra-operative period were significantly higher in the remifentanil group, when compared to the fentanyl group: 8 (2) kPa vs 6.7 (1.5) kPa, % CI difference: - 2.3 kPa to - 0.3 kPa, p = 0.013. Continuous intra-operative opioid administration may blunt vasoconstriction caused by surgical stress and adrenergic responses more than an equi-effective anaesthetic regimen based on smaller-dose bolus opioid administration.

Hemoglobin vesicles reduce hypoxia-related inflammation in critically ischemic hamster flap tissue

OBJECTIVES: The aim of this study was to investigate the effect of a highly viscous, left-shifted hemoglobin vesicle solution (HbV) on the hypoxia-related inflammation and the microcirculation in critically ischemic peripheral tissue. DESIGN: Randomized prospective study. SETTING: University laboratory. SUBJECTS: Twenty-four male golden Syrian hamsters. INTERVENTIONS: Island flaps were dissected from the back skin of anesthetized hamsters for assessment with intravital microscopy. The flap included a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. One hour after completion of the preparation, the animals received an injection of 25% of total blood volume of 0.9% NaCl or NaCl suspended with HbVs at a concentration of 5 g/dL (HbV5) or 10 g/dL (HbV10). MEASUREMENTS AND MAIN RESULTS: Plasma viscosity was increased from 1.32 cP to 1.61 cP and 2.14 cP after the administration of HbV5 and HbV10, respectively (both p < .01). Both HbV solutions raised partial oxygen tension (Clark-type microprobes) in the ischemic tissue from approximately 10 torr to 17 torr (p < .01), which was paralleled by an increase in capillary perfusion by > 200% (p < .01). The 50% increase in macromolecular capillary leakage found over time in the control animals was completely abolished by the HbV solutions (p < .01), which was accompanied by a > 50% (p < .01) reduction in cells immunohistochemically stained for tumor necrosis factor-alpha and interleukin-6 and in leukocyte counts, whereas no such changes were observed in the anatomically perfused, normoxic tissue. CONCLUSIONS: Our study suggests that in critically ischemic, hypoxic peripheral tissue, hypoxia-related inflammation may be reduced by a top-load infusion of HbV solutions. We attributed this effect to a restoration of tissue oxygenation and an increase in plasma viscosity, both of which may have resulted in attenuation of secondary microcirculatory impairments.

Association between elevated brain tissue glycerol levels and poor outcome following severe traumatic brain injury

OBJECT: Glycerol is considered to be a marker of cell membrane degradation and thus cellular lysis. Recently, it has become feasible to measure via microdialysis cerebral extracellular fluid (ECF) glycerol concentrations at the patient's bedside. Therefore the aim of this study was to investigate the ECF concentration and time course of glycerol after severe traumatic brain injury (TBI) and its relationship to patient outcome and other monitoring parameters. METHODS: As soon as possible after injury for up to 4 days, 76 severely head-injured patients were monitored using a microdialysis probe (cerebral glycerol) and a Neurotrend sensor (brain tissue PO2) in uninjured brain tissue confirmed by computerized tomography scanning. The mean brain tissue glycerol concentration in all monitored patients decreased significantly from 206 +/- 31 micromol/L on Day 1 to 9 +/- 3 micromol/L on Day 4 after injury (p < 0.0001). Note, however, that there was no significant difference in the time course between patients with a favorable outcome (Glasgow Outcome Scale [GOS] Scores 4 and 5) and those with an unfavorable outcome (GOS Scores 1-3). Significantly increased glycerol concentrations were observed when brain tissue PO2 was less than 10 mm Hg or when cerebral perfusion pressure was less than 70 mm Hg. CONCLUSIONS: Based on results in the present study one can infer that microdialysate glycerol is a marker of severe tissue damage, as seen immediately after brain injury or during profound tissue hypoxia. Given that brain tissue glycerol levels do not yet add new clinically significant information, however, routine monitoring of this parameter following traumatic brain injury needs further validation.