Hepatocyte Transplantation to the Liver via the Splenic Artery in a Juvenile Large Animal Model.

Hepatocyte transplantation (HcTx) is a promising approach for the treatment of metabolic diseases in newborns and children. The most common application route is the portal vein, which is difficult to access in the newborn. Transfemoral access to the splenic artery for HcTx has been evaluated in adults, with trials suggesting hepatocyte translocation from the spleen to the liver with a reduced risk for thromboembolic complications. Using juvenile Göttingen minipigs, we aimed to evaluate feasibility of hepatocyte transplantation by transfemoral splenic artery catheterization, while providing insight on engraftment, translocation, viability, and thromboembolic complications. Four Göttingen Minipigs weighing 5.6 kg to 12.6 kg were infused with human hepatocytes (two infusions per cycle, 1.00E08 cells per kg body weight). Immunosuppression consisted of tacrolimus and prednisolone. The animals were sacrificed directly after cell infusion (n=2), 2 days (n=1), or 14 days after infusion (n=1). The splenic and portal venous blood flow was controlled via color-coded Doppler sonography. Computed tomography was performed on days 6 and 18 after the first infusion. Tissue samples were stained in search of human hepatocytes. Catheter placement was feasible in all cases without procedure-associated complications. Repetitive cell transplantations were possible without serious adverse effects associated with hepatocyte transplantation. Immunohistochemical staining has proven cell relocation to the portal venous system and liver parenchyma. However, cells were neither present in the liver nor the spleen 18 days after HcTx. Immunological analyses showed a response of the adaptive immune system to the human cells. We show that interventional cell application via the femoral artery is feasible in a juvenile large animal model of HcTx. Moreover, cells are able to pass through the spleen to relocate in the liver after splenic artery infusion. Further studies are necessary to compare this approach with umbilical or transhepatic hepatocyte administration.

Individualized positive end-expiratory pressure in obese patients during general anaesthesia: a randomized controlled clinical trial using electrical impedance tomography.

Background: General anaesthesia leads to atelectasis, reduced end-expiratory lung volume (EELV), and diminished arterial oxygenation in obese patients. We hypothesized that a combination of a recruitment manoeuvre (RM) and individualized positive end-expiratory pressure (PEEP) can avoid these effects. Methods: Patients with a BMI ≥35 kg m -2 undergoing elective laparoscopic surgery were randomly allocated to mechanical ventilation with a tidal volume of 8 ml kg -1 predicted body weight and (i) an RM followed by individualized PEEP titrated using electrical impedance tomography (PEEP IND ) or (ii) no RM and PEEP of 5 cm H 2 O (PEEP 5 ). Gas exchange, regional ventilation distribution, and EELV (multiple breath nitrogen washout method) were determined before, during, and after anaesthesia. The primary end point was the ratio of arterial partial pressure of oxygen to inspiratory oxygen fraction ( P aO 2 / F iO 2 ). Results: For PEEP IND ( n =25) and PEEP 5 ( n =25) arms together, P aO 2 / F iO 2 and EELV decreased by 15 kPa [95% confidence interval (CI) 11-20 kPa, P <0.001] and 1.2 litres (95% CI 0.9-1.6 litres, P <0.001), respectively, after intubation. Mean ( sd ) PEEP IND was 18.5 (5.6) cm H 2 O. In the PEEP IND arm, P aO 2 / F iO 2 before extubation was 23 kPa higher (95% CI 16-29 kPa; P <0.001), EELV was 1.8 litres larger (95% CI 1.5-2.2 litres; P <0.001), driving pressure was 6.7 cm H 2 O lower (95% CI 5.4-7.9 cm H 2 O; P <0.001), and regional ventilation was more equally distributed than for PEEP 5 . After extubation, however, these differences between the arms vanished. Conclusions: In obese patients, an RM and higher PEEP IND restored EELV, regional ventilation distribution, and oxygenation during anaesthesia, but these differences did not persist after extubation. Therefore, lung protection strategies should include the postoperative period. Clinical trial registration: German clinical trials register DRKS00004199, www.who.int/ictrp/network/drks2/en/ .

Computed tomography-based survey of the vascular anatomy of the juvenile Göttingen minipig.

Over the past 50 years, image-guided procedures have been established for a wide range of applications. The development and clinical translation of new treatment regimens necessitate the availability of suitable animal models. The juvenile Göttingen minipig presents a favourable profile as a model for human infants. However, no information can be found regarding the vascular system of juvenile minipigs in the literature. Such information is imperative for planning the accessibility of target structures by catheterization. We present here a complete mapping of the arterial system of the juvenile minipig based on contrast-enhanced computed tomography. Four female animals weighing 6.13 ± 0.72 kg were used for the analyses. Imaging was performed under anaesthesia, and the measurement of the vascular structures was performed independently by four investigators. Our dataset forms a basis for future interventional studies in juvenile minipigs, and enables planning and refinement of future experiments according to the 3R (replacement, reduction and refinement) principles of animal research.

Complications of immunosuppressive agents therapy in transplant patients.

Solid organ transplantation remains the gold standard for the treatment of end-stage organ dysfunction and saves thousands of lives. Besides the progress of surgery, advances in understanding transplant physiology, immunology and the development of immunosuppressive drugs lead to improved short- and long-term survival. Transplantation is offered to an increasing number of patients with higher age and comorbidities. Approximately one third of organ recipients require hospital readmission after transplantation because of a multitude of clinical problems related to immunosuppressive therapy. We review the current knowledge on typical complications associated with immunosuppressants with emphasis on the intensivist's perspective.

Validation study of an interpolation method for calculating whole lung volumes and masses from reduced numbers of CT-images in ponies.

Quantitative computer tomographic analysis (qCTA) is an accurate but time intensive method used to quantify volume, mass and aeration of the lungs. The aim of this study was to validate a time efficient interpolation technique for application of qCTA in ponies. Forty-one thoracic computer tomographic (CT) scans obtained from eight anaesthetised ponies positioned in dorsal recumbency were included. Total lung volume and mass and their distribution into four compartments (non-aerated, poorly aerated, normally aerated and hyperaerated; defined based on the attenuation in Hounsfield Units) were determined for the entire lung from all 5 mm thick CT-images, 59 (55-66) per animal. An interpolation technique validated for use in humans was then applied to calculate qCTA results for lung volumes and masses from only 10, 12, and 14 selected CT-images per scan. The time required for both procedures was recorded. Results were compared statistically using the Bland-Altman approach. The bias ± 2 SD for total lung volume calculated from interpolation of 10, 12, and 14 CT-images was -1.2 ± 5.8%, 0.1 ± 3.5%, and 0.0 ± 2.5%, respectively. The corresponding results for total lung mass were -1.1 ± 5.9%, 0.0 ± 3.5%, and 0.0 ± 3.0%. The average time for analysis of one thoracic CT-scan using the interpolation method was 1.5-2 h compared to 8 h for analysis of all images of one complete thoracic CT-scan. The calculation of pulmonary qCTA data by interpolation from 12 CT-images was applicable for equine lung CT-scans and reduced the time required for analysis by 75%.

[Polytrauma with pelvic fractures and severe thoracic trauma: does the timing of definitive pelvic fracture stabilization affect the clinical course?]. / Polytrauma mit Beckenfraktur und schwerem Thoraxtrauma: Hat der Zeitpunkt der definitiven Beckenfrakturstabilisierung Einfluss auf den klinischen Verlauf?

BACKGROUND: The aim of this study was to investigate the influence of the surgical timing in patients with pelvic fractures and severe chest trauma on the clinical course, especially on postoperative lung function. METHODS: A total of 47 patients were included in a prospective dual observational study. The study investigated the clinical course depending on the time of operation based on the functional lung parameters, SAPS II, SOFA and total hospital stay. RESULTS: The average ISS was 32±6, PTS was 34±11 and TTSS was 9±3 points. The pelvic fractures were stabilized definitively after an average of 7±2 days. The early stabilization correlated significantly with a lower TTSS and SAPS II on admission (p<0.05), shorter time of ventilation (p<0.05) and stay in the intensive care unit (p<0.01) as well as the decreased need for packed red blood cells (p<0.01). CONCLUSIONS: In this study patients with pelvic fractures and thoracic trauma benefited positively from an earlier definitive pelvic fracture stabilization with respect to a shorter time of ventilation and stay in the intensive care unit due to a lower need for red cell concentrates.

Extrapolation from ten sections can make CT-based quantification of lung aeration more practicable.

PURPOSE: Clinical applications of quantitative computed tomography (qCT) in patients with pulmonary opacifications are hindered by the radiation exposure and by the arduous manual image processing. We hypothesized that extrapolation from only ten thoracic CT sections will provide reliable information on the aeration of the entire lung. METHODS: CTs of 72 patients with normal and 85 patients with opacified lungs were studied retrospectively. Volumes and masses of the lung and its differently aerated compartments were obtained from all CT sections. Then only the most cranial and caudal sections and a further eight evenly spaced sections between them were selected. The results from these ten sections were extrapolated to the entire lung. The agreement between both methods was assessed with Bland-Altman plots. RESULTS: Median (range) total lung volume and mass were 3,738 (1,311-6,768) ml and 957 (545-3,019) g, the corresponding bias (limits of agreement) were 26 (-42 to 95) ml and 8 (-21 to 38) g, respectively. The median volumes (range) of differently aerated compartments (percentage of total lung volume) were 1 (0-54)% for the nonaerated, 5 (1-44)% for the poorly aerated, 85 (28-98)% for the normally aerated, and 4 (0-48)% for the hyperaerated subvolume. The agreement between the extrapolated results and those from all CT sections was excellent. All bias values were below 1% of the total lung volume or mass, the limits of agreement never exceeded ± 2%. CONCLUSION: The extrapolation method can reduce radiation exposure and shorten the time required for qCT analysis of lung aeration.

[Peripheral nerve blocks of the lower extremities. Clinical and practical aspects]. / Periphere Nervenblockaden an der unteren Extremität. Klinisch-praktische Aspekte.

Peripheral nerve blocks (PNBs) of the lower extremities are effective techniques for anesthesia and postoperative pain control. So far, these techniques have been used less frequently than PNBs of the upper limb. Nevertheless, growing awareness of complications of neuroaxial techniques, improved equipment and modern techniques for nerve localization have led to an increased use of PNBs of the lower limb. Anesthesiologists should be familiar with the anatomical basics and procedural details of these PNBs. They should also know the typical complications and side-effects and thoroughly inform patients about such potential problems. Continuous PNBs (perineural catheters) allow the benefits of PNBs to be prolonged into the postoperative period. Compared to continuous neuroaxial techniques continuous PNBs are equally effective for pain control but seem to be associated with fewer complications and side-effects.

[Qualitative and quantitative CT analysis of acute pulmonary failure]. / Qualitative und quantitative CT-Analysen beim akuten Lungenversagen.

Since its first application in patients with acute lung injury 25 years ago, computed tomography (CT) has significantly influenced the understanding of the pathophysiology, diagnosis and management of acute lung injury and has become an important diagnostic modality for these patients. The aim of this article is to review important disease-specific aspects of CT acquisition and qualitative and quantitative analyses of CT data. Morphological changes seen on CT and associated functional alterations are discussed. Methods used for the quantification of lung aeration are described and their limitations outlined.

Anterior sciatic nerve block--new landmarks and clinical experience.

BACKGROUND: Anterior sciatic nerve blocks can be complicated by several problems. Pain can be caused by bony contacts and, in obese patients, identification of the landmarks is frequently difficult. METHODS: In a first step, 100 normal anterior-posterior pelvic X-rays were analyzed. The landmarks of the classical anterior approach were drawn on these X-rays and assessed for their sufficiency. Then, in a prospective case study, 200 consecutive patients undergoing total knee replacement were investigated. These patients received femoral and sciatic nerve catheters for postoperative pain management. Using modified anatomical landmarks, sciatic nerve catheters were inserted 5 cm distal from the insertion site of the femoral nerve block perpendicularly in the midline of the lower extremity. This midline connected the insertion site of the femoral nerve catheter to the midpoint between the medial and lateral epicondyle. Correct catheter positioning was verified by magnetic resonance imaging (MRI) in six patients. RESULTS: Evaluation of pelvic X-rays showed that puncture following the classical landmarks pointed in 51% at the lesser trochanter, in 5% medial to the lesser trochanter and in 42% directly at the femur. In the latter patients, location of the sciatic nerve would have been difficult or even impossible. Using our modified anterior approach, the sciatic nerve could be blocked in 196 patients (98%). In nine patients (4.5%) blockade of the posterior femoral cutaneous nerve failed. Vascular puncture happened in 10 (5%) and bony contact in 35 patients (17.5%). Median puncturing depth was 9.5 (7.5-14) cm. Correct sciatic nerve catheter positioning was verified in all patients who underwent MRI. CONCLUSION: Our landmarks for locating the sciatic nerve help to avoid bony contacts and thereby reduce pain during puncture. Our method reliably enabled catheter placement.

Inhalation injury assessed by score does not contribute to the development of acute respiratory distress syndrome in burn victims.

OBJECTIVE: To establish the incidence, mortality, and time of onset of acute respiratory distress syndrome (ARDS) in relation to extent of burn and inhalation injury in patients who required mechanical ventilation. DESIGN: Data about burn and inhalation injury were recorded prospectively whereas ARDS and multiple organ dysfunction were assessed by review of patient charts. SETTING: National burn intensive care unit at Linkoping University Hospital, Sweden (a tertiary referral hospital). PATIENTS: Between 1993 and 1999, we studied all patients with thermal injury (n=553) who required mechanical ventilation for more than two days (n=91). MEASUREMENTS AND RESULTS: Out of the thirty-six burn victims who developed ARDS (40%), 25 (70%) did so early post burn (in less than 6 days). Patients with ARDS had higher multiple organ dysfunction scores (mean 10.5) than those who did not develop ARDS (mean 5.6) (p<0.01). The probable presence of inhalation injury as assessed by an inhalation lung injury score (ILIS) did not contribute to the development of ARDS. Mortality tended to be higher in patients who developed ARDS (14%) compared to those who did not (6%, p=0.2). CONCLUSIONS: In our burn patients the incidence of ARDS was high whereas mortality was low. We found no association between inhalation injury as assessed using the ILIS and development of ARDS. Our data support a multi-factorial origin of ARDS in burn victims as a part of a multiple organ failure event.

Computed tomography--a possible aid in the diagnosis of smoke inhalation injury?

Inhalation injury is an important contributor to morbidity and mortality in burn victims and can trigger acute lung injury and acute respiratory distress syndrome (ARDS) (1-3). Early diagnosis and treatment of inhalation injury are important, but a major problem in planning treatment and evaluating the prognosis has been the lack of consensus about diagnostic criteria (4). Chest radiographs on admission are often non-specific (5, 6), but indicators include indoor fires, facial burns, bronchoscopic findings of soot in the airways, and detection of carbon monoxide or cyanide in the blood (7). Changes in the lungs may be detected by bronchoscopy with biopsy, xenon imaging, or measurement of pulmonary extracellular fluid (4, 5, 8). These methods have, however, been associated with low sensitivity and specificity, as exemplified by the 50% predictive value in the study of Masanes et al. (8). Computed tomographs (CTs) are better than normal chest radiographs in the detection of other pulmonary lesions such as pulmonary contusion (9, 10). The importance of CT scans in patients with ARDS has been reviewed recently (9), but unfortunately there has been no experience of CT in patients with smoke inhalation injury. To our knowledge, there are only two animal studies reporting that smoke inhalation injury can be detected by CT (4, 11); specific changes in human CT scans have not yet been described. Therefore, confronted with a patient with severe respiratory failure after a burn who from the history and physical examination showed the classic risk factors for inhalation injury, we decided to request a CT.

Ventilation by the Open Lung Concept in spite of traumatic lung herniation.

Traumatic herniation of the lung is uncommon. We report a patient suffering from multiple injuries including severe pulmonary contusion and traumatic parasternal lung herniation, who developed acute respiratory distress syndrome. In spite of the lung herniation, we used mechanical ventilation according to the Open Lung Concept. Oxygenation improved rapidly, and early operative stabilization was possible.

[The open lung concept. Clinical application in severe thoracic trauma]. / Das Open Lung Concept. Klinische Anwendung beim schweren Thoraxtrauma.

INTRODUCTION: The pathomorphological substratum of the pulmonary contusion is a parenchymatous hemorrhage followed by interstitial and alveolar edema, finally resulting in a severe damage of the surfactant system. The pathophysiological consequence is an imbalance between ventilation and perfusion, which causes the clinical finding of hypoxia. METHODS: Between December 1997 and December 2000, we treated 32 polytraumatized patients (ISS 43, PTS 32) additionally suffering from severe chest contusion (AIS 5, PTST 14), by ventilation according to the Open Lung Concept (OLC). The initial disturbance of oxygenation was shown by a mean paO2/FIO2-ratio of 134 (96;181) mmHg. The OLC recruits atelectatic lung areas by the application of a defined temporary positive inspiratory pressure (PIP), which is called the "opening pressure". The recruited lung areas were kept open by high total-PEEP. RESULTS: For the recruitment procedure, a mean PIP of 65 (51;65) mbar was required. Recruited alveoli were kept open by a total-PEEP of 22 (20;23) mbar. The paO2/FIO2-ratio increased significantly (P < 0.001) from 134 (96;181) to 522 (433;587) mmHg. After the recruitment procedure, we could reduce PIP and FIO2. In spite of the minimal tidal volumes of 3.5 (3.0;3.9) ml per kg bodyweight by which our patients were ventilated, the levels of oxygenation and normocapnia could be maintained. There were no evidences for side-effects like perfusion impairment. Two patients (6.25%) died of extrapulmonary causes. CONCLUSION: Ventilation according to the OLC seems to be a highly effective treatment of ventilation-perfusion-impairment following pulmonary contusion. Minimal tidal volumes and the low PIP-levels after the recruitment procedure meet the demands of a lung-protective Low-Tidalvolume-Ventilation.

Ras mutations are rare in solitary cold and toxic thyroid nodules.

OBJECTIVE: Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. DESIGN: To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. MEASUREMENTS: Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. RESULTS: Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. CONCLUSION: Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

Biological monitoring of standardized exposure to ethylbenzene: evaluation of a biological tolerance (BAT) value.

The results of standardized 8 h lasting exposures of n = 18 volunteers to ethylbenzene (EthBz) at levels of 25 and 100% of the maximum allowable concentrations at the workplace (MAK) value of 100 ppm as well as the results of field studies are considered to evaluate a biological tolerance (BAT) value for EthBz. On the basis of the relationship between the external and internal exposure a BAT value of 1.5 mg/l has been set for the EthBz concentration in blood as the most sensitive and specific parameter of exposure to this aromatic hydrocarbon. The interpretation of EthBz blood values has to take into account the short half-life of t1/2 = 0.5 +/- 0.08 h in the first hour after the end of exposure in which this aromatic hydrocarbon is eliminated from the blood. The additional determination of the EthBz metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA), respectively, excreted in post shift urine as well as in urine samples at the beginning of the next shift shows good correlations with the external exposure. The biological half-life of MA was calculated to t1/2 = 5.3 +/- 1.1 h. Because the time of sampling can vary the relationship between the levels of MA to PGA the total concentration of the excreted metabolites depends less on this influence and is therefore better suited for monitoring exposed persons. On the basis of the standardized experiments a BAT value has been proposed of 2 g MA plus PGA corrected per gram creatinine. Both BAT values are adjusted to data which result from earlier standardized exposures during 30 min to EthBz under physical activity of 50 watt on a bicycle ergometer.

Linkage of familial euthyroid goiter to the multinodular goiter-1 locus and exclusion of the candidate genes thyroglobulin, thyroperoxidase, and Na+/I- symporter.

Iodine deficiency is the most important etiological factor for euthyroid endemic goiter. However, family and twin pair studies also indicate a genetic predisposition for euthyroid simple goiter. In hypothyroid goiters several molecular defects in the thyroglobulin (TG), thyroperoxidase (TPO), and Na+/I- symporter (NIS) genes have been identified. The TSH receptor with its central role for thyroid function and growth is also a strong candidate gene. Therefore, we investigated a proposita with a relapsing euthyroid goiter and her family, in which several members underwent thyroidectomy for euthyroid goiter. Sequence analysis of the complementary DNA (cDNA) of the TPO and TSH receptor genes revealed several previously reported polymorphisms. As it is not possible to exclude a functional relevance for all polymorphisms, we opted for linkage analysis with microsatellite markers to investigate whether the candidate genes are involved in the pathogenesis of euthyroid goiter. The markers for the genes TG, TPO, and NIS gave two-point and multipoint logarithm of odds score analysis scores that were negative or below 1 for all assumed recombination fractions. As no significant evidence of linkage was found, we conclude that these candidate genes can be excluded as a major cause of the euthyroid goiters in this family. In contrast, we have found evidence for linkage of familial euthyroid goiter to the recently identified locus for familial multinodular nontoxic goiter (MNG-1) on chromosome 14q. The haplotype cosegregates clearly with familial euthyroid goiter. Our results provide the first confirmation for MNG-1 as a locus for nontoxic goiter.

Influence of artificial blood contamination of the analysis of cerebrospinal fluid.

The influence of artificial blood contamination on the quantitative values obtained in the routine examination of the CSF was investigated. On the basis of dilution series from CSF and blood, the correlation between the number of added erythrocytes and the results of leukocyte counts, protein, glucose and IgG estimation was studied. In addition, the influence of the time between CSF sampling and investigation on erythrocytes and leukocytes, IgG, glucose, pH value and ammonia content in CSF contaminated with blood was investigated. The following data relevant to routine examinations resulted: there are linear correlations between the number of erythrocytes artificially added to the CSF and the leukocyte count, the total protein and the IgG content, whereas glucose is unaffected by the artificial admixture of blood. With regard to the time between sampling and investigation of the CSF, it was shown that the IgG values in blood-contaminated and blood-free CSF do not change. A correct IgG estimation is hence possible even several days after lumbar puncture. The cell count decreases exponentially with time, whereas the total protein rises with progressive cytolysis. The glucose values decrease both in native and in artificially blood-contaminated CSF, whereas the pH values rise rapidly, evidently due to release of ammonia and primary amines from the proteins and amino acids present in the CSF.