Donor-derived transplant acceptance-inducing cells in composite tissue allotransplantation.

BACKGROUND: Composite tissue allotransplantation (CTA) is a newly emerging field of transplantation. Immunological research in CTA has been intensified due to the recent clinical success of hand and face transplantation. Establishing immunological tolerance by adoptive transfer of ex vivo cultured tolerance-inducing cell types is of growing interest. Transplant acceptance-inducing cells (TAICs) are a type of deactivated immunoregulatory macrophages. METHODS: A total of 36 allogeneic hind limb transplantations in the rat were performed in six groups. Group A (Lewis (LW) â†’ Brown-Norway (BN)) received Lewis-donor-derived TAICs locally (i.m.). Group B (LW â†’ BN) received Lewis-donor-derived TAICs systemically (i.v.) and group C (Sprague Dawley (Sp-D) â†’ BN) served as a control group receiving Lewis-donor-derived TAICs systemically (i.v.). Groups D (LW â†’ BN), E (LW â†’ BN), and F (BN â†’ BN) also served as control groups with group D receiving no immunosuppression, group E receiving FK506 and prednisolone and group F receiving no immunosuppression with isograft transplantations (BN â†’ BN). The timing of rejection was assessed by clinical observation and histological findings. RESULTS: Rejection of the allogeneic hind limb occurred on average 7.7 days after transplantation in group A and 7.4 days in group B. Rejection was significantly delayed (Log-rank test, p < 0.01) compared to groups C and D, where rejection of the allogeneic hind limb occurred on average 5.8 days and 5.6 days after transplantation. No rejection was seen in groups E and F. CONCLUSION: For the first time, TAICs have been applied in a CTA model and demonstrated a significant immunosuppressive effect. Even though the immunomodulatory effect is relatively modest, the results of this study justify subsequent research on TAIC therapy to improve experimental and clinical outcome after CTA.

[Reduction of the immunological rejection in composite tissue allotransplantation by heat stress preconditioning]. / Reduktion der immunologischen Abstoßungsreaktion in Composite Tissue Allotransplantation durch Hitzeschock-Präkonditionierung.

BACKGROUND: In spite of great advances in the field of composite tissue allotransplantations (CTA), there is still a major need for optimisation in terms of immunosuppression. Heat shock proteins are produced as a reaction of the body during a stress situation. Once elevated, they protect against a second stress and reduce ischaemia-reperfusion injury within transplantations. In the literature the effect of heat shock and HSP70 on rejection after CTA has not been described. The purpose of this experimental study was to examine the effect of heat shock proteins on rejection in a rat model of CTA. Evaluated was the effect of preconditioning by prior heat stress. METHODS AND MATERIALS: Brown Norway rats were systemically heated to a core temperature of 42 °C in order to up-regulate HSP70. The expression of HSP70 in muscle was measured by Western blot analysis and showed a peak 24 h after heat shock. Allogeneic hindlimb transplantations were performed between Brown Norway rats (donor) and Lewis rats (recipients). Group 1 (n=12) was preheated 24 h prior to transplantation. In group 2 (n=12) the transplantation was performed without prior heat shock. Group 3 (n=6) was used as a control group with syngeneic hindlimb transplantations between Lewis rats. Postoperatively the appearance of the transplanted hindlimb was evaluated every 12 h. The beginning of rejection was defined when plantar erythema and foot oedema could be observed at the same time. To verify these discrete signs of rejection, the observation was continued for a further 24 h. In this time erythema and oedema spread over the whole transplanted hindlimb. The rat was sacrificed after specimens of skin and muscle had been taken for histological assessment. RESULTS: The rejection in group 1 (with preconditioning heat shock) began after 4.83±0.44 days, in group 2 (without heat shock) already after 3.88±0.53 days. The difference between these groups was significant because of the small standard deviation (Whitney-Mann U test: p<0.01). CONCLUSION: In our model of allogeneic composite tissue transplantation, a heat shock and subsequent up-regulation of HSP70 led to a significant delay of the immunological rejection. As the graft rejection is an important item influencing the outcome of allogeneic transplantations, these results represent an option to improve the final functional outcome of composite tissue allotransplantations.

Chronic alcohol intake increases the severity of pancreatitis induced by acute alcohol administration, hyperlipidemia and pancreatic duct obstruction in rats.

BACKGROUND: The mechanism of alcoholic pancreatitis is still unknown. It is of special interest why only about 5% of all alcoholics develop an episode of pancreatitis. We evaluated the role of long-term alcohol intake in the pathogenesis of alcoholic pancreatitis in rats. METHODS: To evaluate the effect of long-term alcohol intake, rats were fed either a Lieber-DeCarli control diet (CD) or a Lieber-DeCarli alcohol diet (AD) for 6 weeks. Then, rats were infused over 2 h with either Ringer's solution (CO) or ethanol (E). In additional animals, alcoholic pancreatitis was induced by ethanol combined with hyperlipidemia and temporary pancreatic duct obstruction (EFO). Controls received Ringer's solution combined with hyperlipidemia and temporary pancreatic duct obstruction (RFO). Intravital microscopy (pancreatic perfusion and leukocyte adhesion), alcohol concentrations, amylase, lipase, cholesterine and triglyceride levels in plasma, myeloperoxidase activity and histology were evaluated at different time intervals. RESULTS: In those animals which received the Lieber-DeCarli control diet, capillary perfusion was reduced in the E group and further reduced in the EFO group as compared to the controls (CO, RFO; p < 0.01). Leukocyte adhesion was significantly increased in rats receiving E (p < 0.01), and was further increased in the combination group EFO (p < 0.01). EFO induced histologically evident acute pancreatitis. The additional administration of a long-term alcohol diet further increased microcirculatory disturbances and pancreatic injury significantly (EFO-AD > EFO-CD). CONCLUSIONS: This study shows that alcoholic pancreatitis is induced by the combination of ethanol and individual cofactors. Chronic alcohol abuse intensifies these changes. Therefore, long-term alcohol intake seems to be a major factor in the pathogenesis of alcoholic pancreatitis.

Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer.

Genetic modification of human bone marrow mesenchymal stem cells (MSC) is highly valuable for their exploitation in basic science and therapeutic applications, for example in cancer. We present here a new, fast and easy-to-use method to enrich a functional population of lentiviral (LV)-transduced MSC expressing enhanced green fluorescent protein (eGFP). We replaced the eGFP gene by a fusion gene of puromycin acetyltransferase and eGFP. Upon LV gene transfer and puromycin selection, we quickly obtained a pure transduced MSC population, in which growth, differentiation capacity and migration preferences were not compromised. Furthermore, we are the first to report the migration velocity of MSC among which 30% were moving and velocity of about 15 mum h(-1) was not altered by LV transduction. Manipulated MSC underwent senescence one passage earlier than non-transduced cells, suggesting the use for therapeutic intervention in early passage numbers. Upon tail vein application in nude mice, the majority of LV-transduced MSC could be detected in human orthotopic pancreatic tumor xenografts and to a minor extent in mouse liver, kidney and lung. Together, LV transduction of genes to MSC followed by puromycin selection is a powerful tool for basic research and improves the therapeutic prospects of MSC as vehicles in gene therapy.

Taurine protects from liver injury after warm ischemia in rats: the role of kupffer cells.

BACKGROUND/AIMS: Warm ischemia to liver with subsequent Kupffer cell-dependent pathology is associated with many clinical conditions. Taurine prevents Kupffer cell activation and improves graft survival after experimental cold ischemia and liver transplantation. Thus this study was designed to assess its effects after warm hepatic ischemia. METHODS: The left liver lobe of female Sprague-Dawley rats (170-210 g) underwent 60 min of warm ischemia. Animals were given either intravenous taurine or Ringer's solution 10 min prior to warm ischemia. Transaminases, histology, in vivo microscopy, intercellular adhesion molecules-1 (ICAM-1) expression, TNF-alpha and tissue hydroperoxide were compared between groups using analysis of variance (ANOVA) or ANOVA on ranks as appropriate. RESULTS: Taurine significantly decreased transaminases and improved histologic outcome. Phagocytosis of latex beads, serum TNF-alpha levels and tissue hydroperoxide concentrations were also significantly reduced. Stickers in sinusoids and post-sinusoidal venules significantly decreased. In parallel, both leukocyte infiltration and ICAM-1 expression decreased (p < 0.05), while flow velocity of red blood cells as well as sinusoidal perfusion rate were improved (p < 0.05). CONCLUSION: This study demonstrates that taurine blunts Kupffer cell-dependent hepatic pathology after warm ischemia in vivo via mechanisms including leukocyte-endothelial interaction, microcirculation disturbances and protection against lipid peroxidation.

Do contrast media (iomeprol, gadopentetate dimeglumine) deteriorate ischemia/reperfusion injury of the liver?

BACKGROUND: Hepatic microcirculation is a main determinant of reperfusion injury and graft quality in liver transplantation. One of the important diagnostic procedures to recognize reperfusion failure is contrast-enhanced computed tomography or magnetic resonance imaging. PURPOSE: To examine the additional effect of contrast media (iomeprol and gadopentetate dimeglumine) on hepatic microcirculation and hepatic cellular damage in the phase of early ischemia/reperfusion injury of the rat liver. MATERIAL AND METHODS: The partial warm ischemia-reperfusion injury model of rat liver was used. Microcirculation and leukocyte-endothelium interaction were measured by intravital microscopy. Hepatic cellular damage was indicated by liver enzyme activity in the sera. The evaluation parameters were measured at baseline and at 30, 60, and 90 min after reperfusion. The contrast media (iomeprol group, n = 6; gadopentetate dimeglumine group, n = 6) or Ringer's solution (control group, n = 8) were applied after 30 min of reperfusion. RESULTS: No additional injury to the ischemia/reperfusion injury of the liver after intravenous application of radiographic contrast media was found. Some protective effect was even recorded after application of iodinated contrast media. CONCLUSION: The use of contrast media during diagnostic procedure of the liver seems to be relatively safe, even in the stage of early reperfusion after liver transplantation.

Alterations of tumor and normal tissue of human lung cancer resection specimens after isolation perfusion.

The isolation perfusion model, including transbronchial ventilation of human lung, offers the possibility to study pharmacological interactions under physiological conditions. In view of the increasing importance of targeted therapy of lung diseases, this model of perfusion might attract major interest, particularly, in lung cancer. Our study investigated physiological, histological, and immunohistochemical alterations of lung and tumor tissue during isolated perfusion of lung lobectomy specimens to explore potential limitations of this model. Right after resection, 16 human lung resection specimens for primary lung cancer were isolated, ventilated, and perfused under physiological conditions with a modified Krebs-Henseleit solution over a period of 10, 60, 90, 120, and 240 min. Perfusion pressure, pH, lung weight gain, and histological edema formation were measured continuously before and during perfusion. After perfusion, lung and tumor tissue was investigated by hematoxylin-and-eosin stained sections. Immunohistochemistry of NADH, PECAM-1, angiotensin-converting-enzyme and NF-kappabeta were performed to determine lung tissue viability and changes at the endothelial layer. We found that perfusion up to 120 min could be performed with completely stable physiological conditions. Beyond that time span, edema formation and weight gain of the resection specimen started and were followed by an increase in inspiratory pressure and pulmonary artery pressure. Perfusion of more than 4 h led to a significant edema formation in lung tissue accompanied by loss of viability and significant histological alterations. We conclude that isolated ventilation and perfusion of human lung resections within the setup chosen is reliable for pharmacological studies up to a period of 120 min. Thereafter, edema formation and endothelial damage develop and limit the interpretation and reliability of drug delivery studies.

Bradykinin in ischemia-reperfusion injury of the rat lung.

Recent studies described possibilities to reduce lung damage after intestinal ischemia by application of a selective bradykinin-2 receptor antagonist (HOE 140). In contrast, it has been shown that the preischemic application of bradykinin (BK) reduced ischemic damage of the myocardium. In the present study, to evaluate the effects of BK and HOE 140 in lung ischemia-reperfusion injury we used a standardized in vivo ischemia-reperfusion model of the right rat lung. Ischemia of 60 min was induced by cross-clamping of the right hilus followed by 120 min of reperfusion. During reperfusion, the left hilus was ligated. In Group 1 (n=5), the animals were sham operated without induction of ischemia under ligation of the left lung hilus. Group 2 (n=5) was operated as described, Group 3 (n=5) received 100 microg bradykinin (BK) before reperfusion, Group 4 (n=5) was given a B2-agonist before reperfusion, and Group 5 (n=5) was given 100 microg HOE140/kg body weight before reperfusion. Blood pressure and arterial oxygenation were monitored. As a marker of endothelial damage, angiotensin-converting-enzyme activity (ACE) in serum and RT-PCR of ACE and angiotensin-2 in lung tissue were determined in all groups. Two of the HOE140-treated animals died within 30 min of reperfusion. During reperfusion, significantly higher PaO2 values (P<0.01) have been observed in BK treated animals of Group 3 (214+/-22 mmHg) and sham operated controls of Group 1 (233+/-26 mmHg) compared with Groups 2 (132+/-13 mmHg) and Group 5 (125+/-50 mmHg; P<0.01). Serum ACE activity after reperfusion was significantly lower in Group 1 (3.5+/-0.5 IU/l), Group 3 (3.8+/-1.1 IU/l), and Group 4 (2.2+/-0.5 IU/l; P<0.05) vs. Group 2 (4.8+/-0.9 IU/l), whereas Group 5 (6.2+/-5.4 IU/l) did not differ from Group 2. mRNA expressions of ACE was lower in Group 1 and Group 3 compared with Group 2 (P<0.01). AT-2 mRNA expression did not show any differences between the investigated groups. A significantly lower ACE activity and expression and a significantly higher oxygenation after BK application in Group 3 strongly suggest a positive influence of bradykinin on ischemic preconditioning of the pulmonary endothelium. Positive effects of application of bradykinin-receptor antagonists could not be proved in this study.

Development and evaluation of a training module for the clinical introduction of the da Vinci robotic system in visceral and vascular surgery.

BACKGROUND: With the increasing use of the surgical robotic system in the clinical arena, appropriate training programs and assessment systems need to be established for mastery of this new technology. The authors aimed to design and evaluate a clinic-like training program for the clinical introduction of the da Vinci robotic system in visceral and vascular surgery. METHODS: Four trainees with different surgical levels of experience participated in this study using the da Vinci telemanipulator. Each participant started with an initial evaluation stage composed of standardized visceral and vascular operations (cholecystectomy, gastrotomy, anastomosis of the small intestine, and anastomosis of the aorta) in a porcine model. Then the participants went on to the training stage with the rat model, performing standardized visceral and vascular operations (gastrotomy, anastomosis of the large and small intestines, and anastomosis of the aorta) four times in four rats. The final evaluation stage was again identical to the initial stage. The operative times, the number of complications, and the performance quality of the participants were compared between the two evaluation stages to assess the impact of the training stage on the results. RESULTS: The operative times in the final evaluation stage were considerably shorter than in the initial evaluation stage and, except for cholecystectomies, all the differences reached statistical significance. Also, significantly fewer complications and improved quality for each operation in the final evaluation stage were documented, as compared with their counterparts in the initial evaluation stage. These improvements were recorded at each level of experience. CONCLUSIONS: The presented experimental small and large animal model is a standardized and reproducible training method for robotic surgery that allows evaluation of the surgical performance while shortening and optimizing the learning-curve.

Evaluation of microperfusion disturbances in the transplanted liver after Kupffer cell destruction using GdCl3: an experimental porcine study.

BACKGROUND: Organ function after liver transplantation is determined by ischemia-reperfusion injury. Destruction of Kupffer cells with gadolinium chloride (GdCl3) has been shown to have a possible preventive effect on the extent of this injury, which can be extrapolated by analyzing the distribution of hepatic microperfusion. The aim of this study was to evaluate the protective effect of GdCl3 on disturbances of microperfusion in the transplanted liver. METHODS: Landrace pigs were randomly divided into three groups. In the control group (CG; n=6) a mapping of the native liver was conducted. For mapping, the four hepatic liver lobes were named from right to left with A to D and every lobe was divided into three vertical segments (cranial, medial, and caudal). In each of these 12 areas, microperfusion was quantified using a thermodiffusion probe (TD [mL/100 g/min]). The other two groups were considered as transplanted treated group (TTG; n=10) and transplanted nontreated group (TnTG; n=10). The TTG received an infusion of 20 mg/kg GdCl3 intravenously 24 hours before organ harvesting. Then standardized orthotopic liver transplantation was performed. In TnTG, standardized orthotopic liver transplantation was carried out without prior GdCl3 injection. In the recipients, the microperfusion of transplanted livers were mapped in both TnTG and TTG, in two different time points (1 hour [n=5] and 24 hours (n=5]) after reperfusion. RESULTS: A significant reduction of macrophages in the TTG livers in comparison to the CG and TnTG livers was observed (P<.05). However, the number of macrophages in CG and TnTG livers showed no significant difference (P>.05). Regarding liver microperfusion, in TnTG, a marked heterogeneity was detected in the livers after reperfusion. Significant differences between liver lobes (horizontal planes; P=.032) and vertical layers of intralobar liver parenchyma (P=.029) were observed. The same pattern was seen in TTG livers after reperfusion and a significant difference between horizontal (P=.024) and vertical layers (P=.018) of liver tissue were observed. Comparing intralobar regional flow data between vertical planes 24 hours after reperfusion still showed a prominent variation of hepatic tissue perfusion in TnTG livers (P=.028). Within the same horizontal layers, no significant differences between lobes were measured anymore (P=.16). Contrary to TnTG, in TTG, a homogenous pattern of regional liver tissue perfusion was recorded 24 hours after reperfusion. Comparison of TD data on the liver regions showed no significant microperfusion differences in either horizontal (P=.888) or vertical (P=.841) layers. CONCLUSIONS: Application of GdCl3 resulted in a significant reduction of Kupffer cells. Twenty four hours after transplantation microperfusion showed a homogeneous pattern, which constituted an earlier and better recovery of the transplanted liver. Therefore, destruction of Kupffer cells reduced ischemia-reperfusion injury and seemed to be responsible for the early recovery of microperfusion disturbances and thus for an improvement of graft function.

Intravital microscopy of pulmonary microcirculation after single lung transplantation in pigs.

BACKGROUND: Pulmonary reperfusion injury is a significant risk factor following lung transplantation (LTx). Unfortunately, in vivo observations and quantitative analyses of the pulmonary microcirculation following LTx are technically demanding. METHODS: Pigs, weighing 18 to 22 kg, served as the laboratory animals. The left lung was harvested and preserved using donor aortic vessel segments, the pulmonary artery, and the cuff of the lung veins were extended. After 4 hours of ischemia, the lungs were transplanted by direct connection of the conduits to the left atrial appendage and the left pulmonary artery of the recipient. The lungs were placed extrathoracically and ventilated. The recipient left lung was excluded. With this procedure, mechanical trauma to the lung and moving artefacts were avoided. Intravital microscopic observation became feasible. RESULTS: Following reperfusion, oxygenation of pulmonary venous blood was excellent. However, blood flow distribution was significantly reduced to the transplanted lung compared with the native right recipient lung. Pulmonary vascular resistance was significantly increased, dropping from 3500 to 1000 dynes x s x cm(-5) during reperfusion compared to a value of 500 for the native right lung. The pulmonary microcirculation showed a significant number of no-reflow areas with extremely reduced red blood cell velocities. Greater than 90% of microvessels (<30 microm) showed velocities below 0.1 mm/sec. In conclusion, microvascular injury seems to be a major pathogenic factor for the development reperfusion failure. Quantification of alterations within the microvasculature may shed light on various treatment modalities that reduce perfusion failure.

A novel model for the investigation of orthotopically growing primary and secondary bone tumours using intravital microscopy.

Here is reported the development of an experimental model using intravital microscopy as a tool to orthotopically investigate malignant bone tumours. Although up to 85% of the most frequently occurring malignant solid tumours, such as lung and prostate carcinomas, metastasize into the bone, and despite the knowledge that a tumour's course may be altered by its surrounding tissue, there is no adequate experimental model available enabling the investigation of orthotopically grown bone tumours in vivo. Intravital microscopy is an internationally accepted experimental method, used in various acute and chronic animal models, that enables qualitative and quantitative analysis of the angiogenesis, microcirculation, growth behaviour, etc. of various benign and malignant tissues. Non-invasive investigations of up to several weeks are possible. Additionally, tissue samples can be taken after termination of the in vivo experiments for further ex vivo investigation (histology, immunohistochemistry, molecular biology, etc.), elucidating the mechanisms that underlie the in vivo observations. Severe combined immunodeficient mice were fitted with a cranial window preparation where the calvaria served as the site for orthotopic implantation of the solid human tumours Saos-2 osteosarcoma (primary) and A 549 lung carcinoma and PC-3 prostate carcinoma (secondary). In all preparations, the take rate was 100%. Histological assessment confirmed the data obtained in vivo, showing typical tumour growth with infiltration of the surrounding osseous and soft tissues. This novel model serves as a valuable tool in understanding the biology of primary and secondary bone tumours in physiological and pathophysiological situations, with implications for the most areas of tumour therapy such as chemotherapy, radiation and antiangiogenesis.

Surface contact measurement of electrical cell uncoupling in the mouse heart during ischemia.

Electrical cell uncoupling via gap junction closure is assumed to cause characteristic changes of the passive dielectric spectrum of ischemic heart tissue. In order to find an independent evidence for this assumption, we analysed heart tissue during ischemia, measured the open state of gap junctions by means of dye transfer and correlated this parameter with the time course of the dielectric permittivity. The hearts were pre-ischemically arrested by perfusion with Ringer solution containing 20 mmol/L of potassium (group KCL, n=10). This solution was also used with the addition of two gap junction blockers, either 3 mmol/L heptanol (group HEP, n=4) or 20 micromol/L palmitoleic acid (group PA, n=7). During subsequent ischemia at 21.0+/-0.5 degrees C, we monitored the passive dielectric permittivity spectrum and the spread of dye. After a sigmoidal increase the dielectric permittivity reached an upper plateau at 61+/-22 min of ischemia in KCL, at 45+/-7 min in PA, and already during perfusion at 2+/-1 min in group HEP. At the beginning of ischemia, dye migrated to neighbouring cells in groups KCL and PA but not in HEP. In KCL and PA, the intercellular diffusion of dye stopped after 64+/-26 and 40+/-11 min of ischemia, respectively. Our results suggest that the sigmoidal increase in dielectric permittivity and the reduction of dye diffusion depend on a common mechanism, namely gap junction closure.

Laparoscopic organ retrieval for living donor liver transplantation does not prevent graft injury.

The cause of transplant failure may be difficult to define. However, organ retrieval before preservation and transplantation is an important factor. Organ manipulation during harvesting, which is inevitable with most techniques, leads to injury upon reperfusion including microcirculatory disturbances. Recently, laparoscopic organ retrieval has been successfully performed for human living donor liver transplantation (LDLT). Pneumoperitoneum for laparoscopy changes the pattern of hepatic blood flow. To study the effects of pneumoperitoneum on the graft prior to cold storage, livers from Sprague-Dawley rats underwent pneumoperitoneum with an intra-abdominal pressure of 8 mm Hg for 90 minutes. Subsequently, intravital microscopy was performed to assess intrahepatic microcirculation and transaminases were measured. Serum transaminases increased 1.5-fold compared with sham controls (P < .05). Intrahepatic microcirculation was significantly disturbed immediately after pneumoperitoneum. If this is confirmed in humans, laparoscopic organ retrieval for LDLT would be expected to decrease graft quality and not be beneficial in liver transplantation.

A review of various techniques of orthotopic liver transplantation in the rat.

Orthotopic liver transplantation (OLT) in rat is a demanding procedure, which has become a popular model to investigate various problems. Our aim was to review and analyze the various techniques of experimental OLT in the rat. A review of the literature revealed 30 techniques or technical modifications. Each modification represented a change or a simplification of the reconstruction method of five anatomical structures, which are cornerstones of a successful OLT: the suprahepatic inferior vena cava (SHVC), portal vein (PV), infrahepatic inferior vena cava (IHVC), hepatic artery (HA), and bile duct (BD). SHVC is anastomosed via microsuture or cuff. The PV anastomosis is performed by microsuture, cuff, or a microsuture-temporary splint technique. IHVC is reconstructed by a microsuture, cuff, or microsuture-temporary splint technique. Arterialization has been accomplished via microsuture (aortic segment, celiac segment, or aortic patch), cuff, splint, sleeve, or telescopic method. Nonarterialization of the graft has also been described. Methods for BD reconstruction include pull-through, telescopic, splint, and T-tube. Although a high level of microsurgical skill is the basic requirement in the microsuture technique which provides the most physiological situation and concomitantly reduces thrombosis, it increases anhepatic time compared to the cuff procedure. The learning curve of microsuture techniques is flat; beginners need much practice to become expert. The most physiologic techniques for anastomoses are preferred for long-term survival studies, while the faster techniques are options for short-term survival studies. Each research group must choose techniques according to study defined aims.

Platelet function in acute experimental pancreatitis induced by ischaemia-reperfusion.

BACKGROUND: Ischaemia-reperfusion (IR)-associated microcirculatory changes play a major role in acute post-transplantation pancreatitis. The pathophysiological role of platelets in these events is unknown. The aim of this study was to examine platelet adhesion and function during early reperfusion after pancreatic ischaemia. METHODS: Rats were subjected to warm pancreatic ischaemia by cross-clamping of the pancreatic vessels for 1 h. After 1 h of reperfusion, platelet-endothelium interaction was evaluated after platelet separation and staining by fluorescence microscopy. Amylase levels and pancreatic histology were evaluated 24 h after reperfusion. Animals treated according to an identical protocol, but without ischaemia, served as controls. RESULTS: Mild pancreatitis had developed by 24 h after IR; serum amylase levels were significantly higher than those in control animals. The numbers of adherent platelets in capillaries and venules were significantly increased, and platelet velocity in capillaries was significantly decreased, in the IR group compared with controls. There was significantly more oedema and inflammation in pancreatic tissue after IR. CONCLUSION: Warm ischaemia for 1 h followed by reperfusion for 24 h caused mild pancreatitis in this experimental model. The pancreatic microcirculation was characterized by pronounced platelet-endothelium interaction in capillaries and venules. These results suggest that platelet activation may play an important role in acute post-transplantation pancreatitis.

Monitoring water content of rat lung tissue in vivo using microwave reflectometry.

Measurement of lung water is an important diagnostic means of assessing pulmonary oedema. Water content affects the dielectric spectrum at microwave frequencies, but quantification is still a problem. A new lung tissue model is presented that allows the calculation of water content from dielectric permittivity. The dielectric permittivity of lung tissue was measured by microwave reflectometry using a non-invasive surface probe. During perfusion of rat lungs (n = 22) with blood, injury was induced by interruption of the blood supply for a duration between 0 (control) and 2 h. Water content was assessed from dielectric permittivity using a new mixture formula and was also determined by drying and weighing. The mixture formula allows for the dielectric polarisation of water, dry matter and air in the tissue. A linear correlation was found between total water content determined from dielectric permittivity and that from drying and weighing (y= 1.001x, R2 = 0.8). Lung injury showed an increase in total water content from 80.9 +/- 1.2% (control) to 84.1 +/- 0.9% (p < 0.01). The analysis of dielectric permittivity data at microwave frequencies with the new tissue model is sensitive enough to detect water accumulation produced by lung injury and it can be used to monitor total water content without tissue destruction.

Monitoring of water content and water distribution in ischemic hearts.

We determined water content and water distribution by fitting dielectric spectra of ischemic canine hearts between 5 MHz and 3 GHz with a newly developed model which describes heart cells and subcellular organelles as rotational ellipsoids filled with electrolyte enclosed by an isolating membrane. The fraction of dry material is modelled by spherical particles with a small dielectric permittivity. Free model parameters were water content, cell volume fraction, and the conductivity of the electrolytes. Resulting model parameters were compared to data from tissue desiccation and to conductivity changes produced by protons and lactate ions. We investigated hearts in two states: during ischemia after interruption of blood flow (pure ischemia, PI, n=5) and during ischemia after resuscitation with Tyrode's solution (IAR, n=14). The difference between water content determined by tissue desiccation and by dielectric spectroscopy was less than 0.5%. During 360 min of ischemia, water content in IAR decreased from 85+/-1.6% to 83+/-2.2% and in PI from 80+/-0.8% to 78+/-1.5%. Cellular volume fraction in IAR increased from 0.47+/-0.045 to 0.63+/-0.031 and in PI from 0.62+/-0.014 to 0.73+/-0.013, which is consistent with published morphometric data. After 180 min of ischemia, the increase of the cytosolic conductivity was 0.14+/-0.02 S/m as calculated from the dielectric spectrum and was similar to the conductivity increase which was roughly estimated on the basis of tissue lactate concentration. In conclusion, dielectric spectroscopy combined with our model analysis facilitates the monitoring of water content and distribution by means of nondestructive surface probes.

Characterization of hepatic parenchymous perfusion heterogeneity and regional flow kinetics after porcine liver transplantation.

BACKGROUND: In clinical practice, a heterogeneous hepatic tissue microperfusion (MC) is often observed after liver resection or transplantation (LTx). Nevertheless this hepatic perfusion phenomenon has never been really quantified with respect to its anatomic distribution and time course in detail. The aim of the study was to characterize liver perfusion heterogeneity and local flow kinetics both in the physiological situation and after standardized ischemia and reperfusion using an established model of porcine LTx. METHODS: Regional distribution of hepatic MC in healthy native porcine livers (control group; n = 8) was analyzed in comparison with data derived 60 min, 24 h, and 72 h after porcine LTx (transplantation group; n = 8 each subgroup; cold ischemia time: 5.7 +/- 1.2 h). MC was measured with implanted thermal diffusion electrodes (TD). Flow in hepatic artery and portal vein was continuously detected by ultrasonic probes. For standardization of measurement localizations, porcine liver lobes were divided anatomically into three horizontal layers (cranial, medial, caudal), defining 12 distinct hepatic measurement regions. RESULTS: In the control group, a homogenous liver MC with a mean flow of 81.6 +/- 13.9 ml/100 g/min was detected in all regions. After LTx, a marked MC heterogeneity was noted 60 min after reperfusion. MC rehomogenization was first documented within horizontal liver planes 24 h later. Comparison of MC between planes showed persisting heterogeneity with a significant intralober drop of mean MC in the cranio-caudal direction. Complete MC rehomogenization (both between horizontal and vertical liver planes) was detected 72 h after reperfusion. Still, an overall reduction of mean liver perfusion by about 15% was existent. CONCLUSIONS: A homogenous tissue perfusion was observed in healthy porcine livers. In contrast, marked heterogeneity of hepatic MC was detected after LTx. Heterogeneity presents as a very dynamic and temporary phenomenon. Early horizontal flow rehomogenization and reconstitution of normal blood flow, particularly primarily in the cranial liver layers, appear to be characteristic features during early flow reconstitution after postischemic reperfusion. Due to heterogeneity and time-dependent flow dynamics, measurement of MC volumes at single hepatic regions may not always allow a valid characterization of liver perfusion quality during the first 24 h after postischemic reperfusion.

In vivo assessment of angioarchitecture and microcirculation in experimental liver cancer: a new model in rats.

The rising incidence of unresectable hepatocellular malignancies remains a therapeutic challenge. Little is known about the mechanisms of angiogenesis and immunological aspects of liver tumor vessels. The aim of this study was to investigate hepatic and tumor microcirculation and leukocyte-endothelium interaction by means of intravital microscopy in a rat model of hepatocellular carcinoma. Off-line analysis showed that the angioarchitecture as well as blood flow velocity in liver cancer is heterogeneous. The leukocyte-endothelium interaction is significantly reduced compared to normal liver tissue. The data suggest that the main mechanism is a reduced expression of adhesion molecules demonstrating an effective immune escape mechanism of this tumor. The model represents a useful experimental tool to explore angiogenesis inhibition or immunological therapeutic strategies in experimental liver cancer.