Fiber optical spatial filter anemometry--intravital measurement of red blood flow velocity (RBCV) in the microcirculation.

The fiberoptical spatial filter anemometry (SFA) is a common technique based on an optical grid to measure the velocity of corpuscular components in a multiphase flow, e.g. in the microvessels. The technical innovation is the analysis of flow velocities using an optical grid sensor and frequency analysis by Fast Fourier Transformation (FFT). This study describes a non-invasive, on-line technique to measure RBCV in the microcirculation. The sensor's validity was proven by in vitro measurements using a rotation disk of an exactly defined velocity with a correlation coefficient of 0.99967. For validation of RBCV measurements in the microcirculation in vivo, the setup was adapted to an intravital microscope. RBCV was measured in arterioles, capillaries, and postcapillary venules ranging from 8-140 microm diameter. As reference method for velocity measurements a computer assisted imaging system was used to measure the RBC-velocity in the identical vessels by frame to frame analysis. Both methods revealed a high significant correlation using transillumination technique for capillaries (r=0.986, p<0.001) and venules (r=0.952, p<0.001) as well as epiillumination technique (capillaries r=0.939, venules r=0.975, p<0.001).

Recipient treatment with L-arginine attenuates donor lung injury associated with hemorrhagic shock.

BACKGROUND: Organ donors are frequently trauma victims, but the impact of donor hemorrhagic shock and resuscitation (HSR) on pulmonary graft function has not been assessed. L-arginine treatment during reperfusion increases the production of endothelial nitric oxide and thus ameliorates ischemia-reperfusion injury. Objective of the present porcine study was to investigate the effect of donor hemorrhage on pulmonary graft function and potential beneficial effects of L-arginine administration. METHODS: In the control-group (n=6), lungs were harvested from donors without hypotensive periods. In the HSR-group (n=6) and HSR-Arg-group (n=6), donors were subjected to hemorrhagic shock (40% blood shed) and resuscitation before harvest. Left lungs were transplanted after hypothermic preservation of 18 hr, and graft function was observed for 6 hr after reperfusion. Recipients in the HSR-Arg-group received a bolus of L-arginine (50 mg/kg BW) intravenously 5 min before reperfusion followed by a continuous intravenous administration of L-arginine 200 mg/kg BW for 2 hr. Tissue specimens and bronchoalveolar lavage fluid were obtained at the end of the observation period. RESULTS: Donor lung function did not differ between study groups. Compared with the control group, pulmonary graft gas exchange was significantly impaired in the HSR-group. Graft function in the HSR-Arg-group did not differ from control organs. Neutrophil fraction, protein content, and malondialdehyde levels in the bronchoalveolar lavage fluid in the HSR-group were higher compared with control and HSR-Arg-Group. CONCLUSION: Although fulfilling ideal donor criteria, pulmonary graft function of lungs harvested from donors subjected to HSR is impaired, but improves significantly when l-arginine is administered during reperfusion.

Resuscitation with recombinant hemoglobin rHb2.0 in a rodent model of hemorrhagic shock.

BACKGROUND: Hemoglobin solutions combine volume effect, oxygen-carrying capacity, and vasoactive properties, the latter facilitating restoration of global hemodynamics but endangering microvascular resuscitation. Hemoglobin-evoked vasoconstriction probably is due to nitric oxide scavenging, which can be reduced by genetic modifications of the heme pocket. This study compares resuscitation with a nonhemoglobin colloid and two recombinant hemoglobin solutions with wild-type and reduced nitric oxide-scavenging capacity. METHODS: Twenty-seven awake Syrian golden hamsters fitted with dorsal skinfold chambers underwent a 30 min-hemorrhagic shock (mean arterial pressure [MAP] 30-35 mmHg) and resuscitation with shed blood volume of either 6% dextran 60 (Biophausia, Uppsala, Sweden), recombinant hemoglobin 1.1 (rHb1.1; wild-type nitric oxide-scavenging capacity; 10 g/dl), or recombinant hemoglobin 2.0 (rHb2.0; reduced nitric oxide-scavenging capacity; 10 g/dl; both Baxter Healthcare, Boulder, CO). Macrohemodynamic and laboratory parameters were assessed; microvascular parameters in the skinfold chamber were analyzed by intravital microscopy. RESULTS: Hemorrhagic shock reduced functional capillary density (FCD) by 70% and caused significant metabolic acidosis. Colloid resuscitation led to incomplete recovery of MAP and FCD. Infusion of rHb1.1 completely restored MAP but not FCD, with the smallest arteriolar diameters found in this group. FCD was restored best by resuscitation with rHb2.0, although MAP was lower than in rHb1.1-treated animals. Metabolic acidosis was resolved by both hemoglobin solutions, but not by dextran. CONCLUSION: After resuscitation with rHb1.1, arteriolar vasoconstriction quickly restored MAP, but this was achieved at the expense of FCD. In contrast, after resuscitation with rHb2.0, the recovery of MAP could be translated into a significantly improved FCD.

Effects of Diaspirin Crosslinked Hemoglobin (DCLHb) on microcirculation and local tissue pO2 of striated skin muscle following resuscitation from hemorrhagic shock.

The hemoglobin based oxygen carrier (HBOC) Diaspirin Crosslinked Hemoglobin (DCLHb) has been developed to substitute not only the blood volume, but also to restore the oxygen-carrying properties of blood during hemorrhagic shock. However, it has been suggested that HBOCs may enhance the formation of free oxygen radicals through the release of free iron ions via the Haber-Weiss reaction. The aim of this study was to investigate the effects of DCLHb on the microcirculation, leukocyte-endothelial cell interaction and local tissue oxygenation in striated skin muscle of Syrian golden hamsters during and after resuscitation from hemorrhagic shock. In particular we focused on the local tissue oxygenation after resuscitation with DCLHb (hemoglobin content 10 g%) compared to resuscitation using autologous blood diluted to a hemoglobin content of 10 g%. Hemorrhagic shock was induced for 45 minutes by bleeding the animals at a rate of 33 ml/kg BW maintaining a mean arterial pressure of 30 +/- 5 mmHg. Animals were resuscitated either with 33 ml/kg BW 6% Dextran-60.000 or with 10 g% DCLHb. The control group received shed blood diluted with Ringers to a hemoglobin content of 10 g%. Intravital microscopy was used for investigation of the microcirculatory parameters and a multiwire platinum surface electrode for measurement of local tissue pO2 in striated skin muscle in the dorsal skinfold chamber of Syrian golden hamsters. Resuscitation from hemorrhagic shock with 10 g% AUB revealed significant increase of leukocytes rolling in postcapillary venules at 30 to 120 minutes after resuscitation compared to baseline values. DCLHb turned out to reduce the number of firmly adherent leukocytes after resuscitation compared to 10 g% AUB. Microvascular permeability as an indicator for functional endothelial integrity revealed no significant differences between the groups. DCLHb and 10 g% AUB led to a significant increase in local tissue oxygenation after resuscitation from hemorrhagic shock. However, 10 g% AUB turned out to be most effective to restore the local tissue pO2 compared to Dx-60. Our findings indicate that DCLHb restores microvascular perfusion after critical hemorrhagic shock as efficient as Dx-60 and 10 g% AUB. The absence of enhanced leukocyte-endothelium interaction after resuscitation with DCLHb implies that this HBOC does not exacerbate formation of oxygen free radicals during reperfusion. DCLHb effectively increases local tissue pO2 after resuscitation from hemorrhagic shock; however, not as effectively as 10 g% AUB.

Intermittent capillary perfusion in rat pancreas grafts following short- and long-term preservation in University of Wisconsin solution.

In pancreas transplantation (PTx), ischemia/reperfusion-induced deterioration of graft-microcirculation is accompanied by alterations of intermittent capillary perfusion (IP; alternating cessation and resumption of capillary blood flow) is known to counteract malperfusion. Incidence and effectiveness of IP following short- versus long-term preservation of pancreas grafts with University of Wisconsin (UW) solution has not been examined so far. PTx was performed in Lewis rats following 2-h or 18-h preservation in UW solution. Using intravital fluorescence microscopy, functional capillary density (FCD), red blood cell (RBC) velocity, IP-incidence and -frequency were analyzed. Laser Doppler flowmetry allowed for the determination of erythrocyte flux and velocity. Measurements were performed at 30, 60 and 120 min after reperfusion. Nontransplanted animals served as controls. FCD, RBC-velocity and -flux remained unchanged in the 2-h group. IP was encountered in 87% of all observation areas at 120 min. After 18-h ischemia, FCD was significantly reduced, which was paralleled by a 50% incidence of IP at 120 min. Tissue edema and leukocyte infiltration in pancreas grafts following 18-h preservation were significantly enhanced. Therefore, IP is an important mechanism aimed at improving microcirculation and UW solution is suitable to preserve vasomotion-activities enabling long-term preservation in a pancreas graft.

Synergistic effects of brain death and liver steatosis on the hepatic microcirculation.

BACKGROUND: The routine transplantation of steatotic livers could potentially mitigate the donor shortage, but so far is associated with a high rate of graft dysfunction. Steatosis and brain death have been perceived as independent risk factors, but they may synergistically target the hepatic microcirculation. This study compares the effects of brain death on the microcirculation of steatotic and normal livers. METHODS: Brain death was induced in obese and lean Zucker rats. Lean and obese sham-operated animals served as controls. Liver microcirculation was investigated using intravital fluorescence microscopy. Serum liver enzyme and reduced glutathione, expression of P-selectin, ICAM-1 and VCAM-1 mRNA in the liver were determined. The ultrastructural alterations were compared by electron microscopy. RESULTS: In nonbrain-dead animals, liver steatosis was associated with smaller sinusoidal diameters, but did not impair sinusoidal perfusion. During brain death, sinusoidal diameter and perfusion were reduced in normal and, to a greater extent, in steatotic livers. Also, more leukocytes were recruited to the microvasculature of steatotic livers than to normal livers in brain-dead state. The highest liver enzyme activities and the lowest hepatic GSH concentrations were measured in brain-dead animals with steatotic livers; only in these organs was endothelial cell swelling regularly observed. In brain-dead state, only the P-selectin mRNA expression was increased in steatotic livers as compared to normal livers. CONCLUSIONS: Brain death amplifies the adverse effects of steatosis on the hepatic microcirculation. Our results underline the need for therapeutic intervention in brain-dead state when steatotic livers are to be used for transplantation.

Effect of prostaglandin I2 analogues on left ventricular diastolic function in vivo.

The prostaglandin I2 analogues epoprostenol and iloprost increase left ventricular contractility. Therefore, we hypothesize that the prostaglandin I2 analogues epoprostenol and iloprost improve also left ventricular diastolic function. To test this hypothesis, the effects of epoprostenol and iloprost on left ventricular diastolic function were assessed in vivo and compared to two vasodilators sodium nitroprusside and adenosine, not formerly associated with changes of left ventricular contractility. Eleven pigs (25.9+/-2.8 kg, balanced anaesthesia) were exposed to the short-acting intravenous vasodilators sodium nitroprusside, adenosine and epoprostenol in a randomized cross over design. The long-acting iloprost was administered at the end of the protocol. The drugs are titrated to achieve a 25% reduction of diastolic aortic pressure. Active isovolumic relaxation properties of the left ventricle were assessed by the maximum velocity of left ventricular pressure drop. Passive phase of relaxation and filling was assessed by the determination of end diastolic compliance during a preload reduction manoeuvre. The maximum velocity of left ventricular pressure drop worsened during the infusion of sodium nitroprusside (baseline: -1950; sodium nitroprusside: -1293 mm Hg/s, p<0.05, Wilcoxon signed rank test versus vs. baseline) and adenosine (baseline: -2015; adenosine: -1345 mm Hg/s, p<0.05), but remained stable during the infusion of the prostaglandins (baseline: -1943; epoprostenol: -1785 mm Hg/s; baseline: -2042; iloprost: -1923 mm Hg/s). End diastolic compliance was not altered significantly by any vasodilator. Interstitial myocardial cAMP increased during the infusion of epoprostenol (7.60 to 13.87 fmol/ml, p<0.05) and tended to increase during the infusion of iloprost (7.56 to 11.66 fmol/ml, p=0.21). The prostaglandin I(2) analogues epoprostenol and iloprost preserved the early phase of active isovolumic relaxation, presumably mediated by myocardial cAMP, whereas sodium nitroprusside and adenosine impaired early active isovolumic relaxation. Passive relaxation and filling properties remained stable during the infusion of each applied vasodilator in the intact left ventricle in vivo.

Mycoplasma haemocanis infection--a kennel disease?

Mycoplasma haemocanis (formerly Haemobartonella canis) is a red blood cell parasite that causes disease mainly in immunosuppressed and splenectomized dogs. Clinical outbreak of the disease resulted in failure of a large experimental project. We aimed to identify whether M. haemocanis has increased prevalence in kennel-raised dogs. In a prospective study, we compared the prevalence of M. haemocanis in whole blood (anti-coagulated by use of EDTA) collected from pet dogs (University of Illinois, Urbana Champaign, Ill.; n = 60) with that in blood from dogs raised in three distinct kennels in western Europe (WE; n = 23), eastern Europe (EE; n = 20), and North America (NA; n = 20). Screening included antibody testing and microscopy of blood smears. The presence of M. haemocanis was identified using a polymerase chain reaction (PCR) assay for specific DNA of the organism. None of the pet dogs (0%) was test positive for M. haemocanis DNA. Mycoplasma haemocanis was found in dogs tested at all of the kennels. Infection rate in the three kennels was 30, 35, and 87%, respectively (all P < 0.001 versus control, chi2-test). Latent infection with M. haemocanis was not a single observation in kennel-raised dogs. Prevalence may be higher than that in a pet dog population. The potential exists for these latent infections to adversely affect or confound research results.

Hypertonic fluid resuscitation from subarachnoid hemorrhage in rats.

OBJECTIVE: Increased intracranial pressure (ICP) and decreased cerebral blood flow leading to global cerebral ischemia are the primary causes of death after severe subarachnoid hemorrhage (SAH). Hypertonic saline has been demonstrated to exert neuroprotective properties after traumatic brain injury by osmotic mobilization of parenchymal water and improvement of microcirculation. We used a rat model to investigate the effects of hypertonic fluid resuscitation after SAH on ICP, cerebral blood flow, body weight, neurological recovery, and morphological damage. METHODS: Sixty rats were subjected to SAH induced by an endovascular filament. ICP and local cerebral blood flow were recorded continuously. Animals were assigned to three groups: 1) NaCl 0.9%; 2) NaCl 7.5% (4 ml/kg); and 3) NaCl 7.5% plus 6% dextran 70 (4 ml/kg) given 30 minutes after SAH. Body weight and neurological deficits were assessed daily. Morphological damage was evaluated on Day 7. RESULTS: SAH resulted in an immediate increase of ICP to approximately 60 mm Hg initially, and then to approximately 30 mm Hg for the next 90 minutes. Although NaCl 7.5% alone and in combination with dextran led to an immediate, significant, and lasting decrease of ICP to 15 to 20 mm Hg, only the combined therapy significantly increased body weight and improved neurological recovery. Furthermore, the group that received combined therapy exhibited significantly more surviving neurons in hippocampus, cortex, caudoputamen, and cerebellum. Mortality was reduced nonsignificantly, from approximately 65% in groups I and II to 35% in Group III. CONCLUSION: Treatment with NaCl 7.5% plus 6% dextran 70 is significantly effective for reducing the initial harmful sequelae of SAH. The regimen resulted in lowered ICP, improved neurological recovery, and less morphological damage after SAH in the rat.

Influence of orthopedic particulate biomaterials on inflammation and synovial microcirculation in the murine knee joint.

The purpose of the present study was to examine changes in the synovial microcirculation as well as synovial tissue responses to exposure to titanium, polymethylmethacrylate (PMMA), ceramic (Al(2)O(3)), cobalt-chromium alloy (Co-Cr), and polyethylene (PE) particles in an in vivo model. The particulate biomaterials were injected into the left knee joint of female Balb/c mice and assessment of the synovial microcirculation using intravital fluorescence microscopy as well as histological evaluation of the synovial tissue response were performed on day 7 after particle administration. Intravital microscopic measurements revealed that all tested biomaterials caused significantly (p < 0.05) enhanced leukocyte-endothelial cell interactions and an increase of functional capillary density compared to controls. In the histological examination PMMA, Al(2)O(3), PE, and Co-Cr particles provoked significantly (p < 0.05) enhanced inflammatory tissue responses in comparison to tissue from control animals. Titanium particles showed significantly (p < 0.05) less leukocyte-endothelial cell interactions than the other particulate biomaterials and caused significantly (p < 0.05) minor membrane thickening compared to PE and PMMA particles. In conclusion, all tested particulate biomaterials were capable of inducing inflammatory responses in the present study. Our data suggest that titanium particles may cause less leukocyte activation and inflammatory tissue responses than other particulate biomaterials used in total joint arthroplasty.

A computer model of fractal myocardial perfusion heterogeneity to elucidate mechanisms of changes in critical coronary stenosis and hypotension.

Critical coronary stenosis (critical CS) alone does not lead to an alteration of fractal dimension (D) under resting conditions in a pig model, indicating undisturbed local myocardial perfusion. If critical CS is combined with hypovolemic anemia the resulting hypotension leads to a significant decline of D. The mechanisms involved in this phenomenon have not yet been elucidated. A computer program was developed enabling calculation of D for normal vascular trees, for single vessel coronary stenosis (CS), and for CS in combination with reduced coronary perfusion pressure (CPP). The values of D obtained by the computer program were compared to those available from an existing animal study to confirm that changes of D can largely be explained by changes of arterial branching pattern simulated by the computer program. Using our computer model, D was 1.15+/-0.06 in normal vascular trees. Third branch critical CS did not alter (1.14+/-0.06; n.s.), whereas critical CS combined with a reduction of CPP to 40 mmHg reduced D (1.07+/-0.03; P < 0.05). These data are comparable to those obtained in the animal study, and therefore show that alterations of vessel diameter and regional blood flow can largely explain changes of fractal dimension during critical CS and hypotension while changes of functional myocardial parameters might play a minor role.

Continuous infusion of nitroglycerin improves pulmonary graft function of non-heart-beating donor lungs.

BACKGROUND: The warm ischemic period of lungs harvested from a non-heart-beating donor (NHBD) results in an increased ischemia-reperfusion injury after transplantation. The intravenous application of nitroglycerin (NTG), a nitric oxide (NO) donor, proved to be beneficial during reperfusion of lung grafts from heart-beating donors. The objective of the present study was to investigate the effect of nitroglycerin on ischemia-reperfusion injury after transplantation of long-term preserved NHBD-lungs. METHODS: Sixteen pigs (body weight, 20-30 kg) underwent left lung transplantation. In the control group (n=5), lungs were flushed (Perfadex, 60 mL/kg) and harvested immediately after cardiac arrest. In the NHBD group (n=5) and the NHBD-NTG group (n=6), lungs were flushed 90 min (warm ischemia) after cardiac arrest. After a total ischemia time of 19 hr, lungs were reperfused and graft function was observed for 5 hr. Recipient animals in the NHBD-NTG group received 2 microg/kg/min of NTG administered intravenously during the observation period starting 5 min before reperfusion. Tissue specimens and bronchoalveolar lavage fluid (BALF) were obtained at the end of the observation period. RESULTS: Compared with the control group, pulmonary gas exchange was significantly impaired in the NHBD group, whereas graft function in the NHBD-NTG group did not change. Leukocyte fraction and protein concentration in the BALF and histologic alteration of the NHBD-NTG group were not different from controls. CONCLUSIONS: Continuous infusion of NTG in the early reperfusion period improves pulmonary graft function of NHBD lungs after long-term preservation. The administration of an NO donor during reperfusion may favor the use of NHBD lungs to alleviate the critical organ shortage in lung transplantation.

Recombinant human hemoglobin with reduced nitric oxide-scavenging capacity restores effectively pancreatic microcirculatory disorders in hemorrhagic shock.

BACKGROUND: Scavenging of nitric oxide by hemoglobin-based oxygen carriers could aggravate microcirculatory failure in splanchnic organs after hemorrhagic shock as a consequence of vasoconstrictive side effects. The aim of this study was to compare the effects of two recombinant human hemoglobin solutions, a second-generation product bearing reduced nitric oxide-scavenging properties (rHb2.0) due to site directed mutagenesis of the heme pocket and a first-generation recombinant hemoglobin (rHb1.1) with scavenging capacity similar to native hemoglobin, on the pancreatic microcirculation after hemorrhagic shock. METHODS: Twenty-eight pentobarbital-anesthetized rats were bled to a mean arterial pressure of 40 mmHg and maintained at this level for 1 h. Using an intravital microscope, the length of erythrocyte-perfused pancreatic capillaries per observation area (functional capillary density) were measured in animals resuscitated by volumes of hydroxyethyl starch, rHb1.1, or rHb2.0 equivalent to the shed blood volume. Animals without shock induction served as control. RESULTS: As compared with control (438 +/- 10 cm(-1)), animals treated with hydroxyethyl starch (315 +/- 44 cm(-1)) and rHb1.1 (288 +/- 67 cm(-1)) showed a significant reduction of functional capillary density after 2 h of resuscitation. rHb2.0 was able to restore functional capillary density (410 +/- 42 cm(-1)) and mean arterial pressure to baseline values. CONCLUSION: rHb2.0 was effectively able to restore pancreatic microcirculation after hemorrhagic shock. This may be related to the compound's effective lack of nitric oxide-scavenging properties. This hemoglobin solution or ones similar to it might be uniquely valuable for resuscitation from hemorrhagic shock.

Intravenous administration of glutathione protects parenchymal and non-parenchymal liver cells against reperfusion injury following rat liver transplantation.

AIM: To investigated the effects of intravenous administration of the antioxidant glutathione (GSH) on reperfusion injury following liver transplantation. METHODS: Livers of male Lewis rats were transplanted after 24 h of hypothermic preservation in University of Wisconsin solution in a syngeneic setting. During a 2-h reperfusion period either saline (controls, n=8) or GSH (50 or 100 micromol/(h/kg), n=5 each) was continuously administered via the jugular vein. RESULTS: Two hours after starting reperfusion plasma ALT increased to 1457+/-281 U/L (mean+/-SE) in controls but to only 908+/-187 U/L (P<0.05) in animals treated with 100 microGSH/(h/kg). No protection was conveyed by 50 micromol GSH(h/kg). Cytoprotection was confirmed by morphological findings on electron microscopy: GSH treatment prevented detachment of sinusoidal endothelial cells (SEC) as well as loss of microvilli and mitochondrial swelling of hepatocytes. Accordingly, postischemic bile flow increased 2-fold. Intravital fluorescence microscopy revealed a nearly complete restoration of sinusoidal blood flow and a significant reduction of leukocyte adherence to sinusoids and postsinusoidal venules. Following infusion of 50 micromol and 100 micromol GSH/(h/kg), plasma GSH increased to 65+/-7 mol/L and 97+/-18 mol/L, but to only 20+/-3 mol/L in untreated recipients. Furthermore, plasma glutathione disulfide (GSSG) increased to 7.5+/-1.0 mol/L in animals treated with 100 micro(h/kg) GSH but did not raise levels of untreated controls (1.8+/-0.5 mol/L) following infusion of 50 microGSH/(h/kg) (2.2+/-0.2 mol/L). CONCLUSION: Plasma GSH levels above a critical level may act as a "sink" for ROS produced in the hepatic vasculature during reperfusion of liver grafts. Therefore, GSH can be considered a candidate antioxidant for the prevention of reperfusion injury after liver transplantation, in particular since it has a low toxicity in humans.

Long-term anaesthesia using inhalatory isoflurane in different strains of mice-the haemodynamic effects.

The aim of this study was to establish a simple and safe method of anaesthesia for intravital microcirculatory observations in small laboratory animals. The usefulness of isoflurane inhalation anaesthesia has been investigated in different strains of mice commonly used in experimental medicine. These were the hairless (hr/hr, n = 12), the BALB/c (n = 12) and the nude mouse (nu/nu, n = 3). Anaesthesia was maintained by mask inhalation of isoflurane vaporized at concentrations of up to 4% in the induction phase, at 1.5% during acute surgical procedures and at 0.8-1.3% during prolonged experimental observations. Isoflurane was vapoured in a N(2)O/O(2) mixture and saturated with 32-36% F(i)O(2). During observations the body temperature was kept constant at 37 degrees C. The tail artery was cannulated for monitoring of mean arterial blood pressure (MAP) and heart rate (HR). To maintain the body fluid balance, isotonic saline was administered at a constant rate of 0.2 ml/h. Arterial blood samples were drawn for blood-gas analysis at the end of the experiments. All animals survived the anaesthesia protocol lasting between 3 and 6.5 h. During isoflurane inhalation, no breathing complications or changes in systemic circulatory parameters were observed. Mean values of MAP and HR were 79+/- 3 mmHg and 486+/- 13 min(-1), respectively, over the entire observation period. A moderate acidosis was recorded in animals under isoflurane anaesthesia, with alterations of arterial blood pH, p(a)O(2) and pCO(2) values (7.29+/- 0.06, 130+/- 19 mmHg and 35.6+/- 4.7 mmHg, respectively). In conclusion, inhalation anaesthesia with isoflurane is useful for experimental studies in the mouse due to (1) the simplicity of administration of the anaesthetic, (2) the rapid induction of anaesthesia, (3) easy control of the depth of anaesthesia, (4) the low percentage of complications, and (5) stable MAP and HR during observations lasting several hours. The proposed technique is especially suitable for observations of the microcirculation under intravital fluorescence microscopy.

Dynamic intravital fluorescence microscopy--a novel method for the assessment of microvascular permeability in acute pancreatitis.

Edema formation is the first manifestation of acute pancreatitis. Microcirculatory derangements like leukocyte-endothelial cell interaction and perfusion failure result in enhancement of microvascular permeability to large molecules playing a pivotal role in the progression of the acutely altered pancreatic tissue. Due to the lack of suitable methods the crucial mechanisms of enhanced permeability in vivo are not very well investigated. Sprague-Dawley rats were randomly assigned to three groups: (a) sham operated animals with normal pancreas, (b) the pancreatitis group induced by 60 min temporary occlusion of the arterial supply followed by reperfusion and (c) the histamine group in which the pancreas was superfused with 10(-5)M histamine. The pharmacokinetics of tetramethylrhodamine-labelled BSA in the intravital microscopic images of a capillary network of the pancreas were densitometrically quantified over 20 min. From these data the effective microvascular permeability was calculated taking also into account morphology of microvessels, elimination rate of the tracer from the intravascular space and capillary microhematocrit. In addition macromolecular leakage of gold-labelled BSA was investigated by electron microscopy. Microvascular permeability was 0.10 +/- 0.02 x 10(-7) cm/s, 0.49 +/- 0.04 x 10(-7) cm/s and 1.21 +/- 0.29 x 10(-7) cm/s for control, ischemia and histamine group, respectively (P < 0.05 ischemia, histamine vs. control and ischemia vs. histamine). Electron microscopy revealed albumin extravasation in the last two groups. We established a technique allowing to quantify microvascular permeability in pancreatic tissue by dynamic intravital microscopy being independent of the investigator. This technique enabling accurate pathophysiologic characterisation in terms of edema formation can form the basis for evaluating in the future novel treatment strategies directed against acute pancreatitis.

Glutathione protects the rat liver against reperfusion injury after prolonged warm ischemia.

OBJECTIVE: To evaluate the potential of postischemic intravenous infusion of the endogenous antioxidant glutathione (GSH) to protect the liver from reperfusion injury following prolonged warm ischemia. BACKGROUND DATA: The release of reactive oxygen species (ROS) by activated Kupffer cells (KC) and leukocytes causes reperfusion injury of the liver after warm ischemia. Therefore, safe and cost-effective antioxidant strategies would appear a promising approach to prevent hepatic reperfusion injury during liver resection, but need to be developed. METHODS: Livers of male Lewis rats were subjected to 60, 90, or 120 minutes of normothermic ischemia. During a 120 minutes reperfusion period either GSH (50, 100 or 200 micromol/h/kg; n= 6-8) or saline (n= 8) was continuously administered via the jugular vein. RESULTS: Postischemic GSH treatment significantly prevented necrotic injury to hepatocytes as indicated by a 50-60% reduction of serum ALT and AST. After 1 hour of ischemia and 2 hours of reperfusion apoptotic hepatocytes were rare (0.50 +/- 0.10%; mean +/- SD) and not different in GSH-treated animals (0.65 +/- 0.20%). GSH (200 micromol GSH/h/kg) improved survival following 2 hours of ischemia (6 of 9 versus 3 of 9 rats; P < 0.05). Intravital fluorescence microscopy revealed a nearly complete restoration of sinusoidal blood flow. This was paralleled by a reduction of leukocyte adherence to sinusoids and postsinusoidal venules. Intravenous GSH administration resulted in a 10- to 40-fold increase of plasma GSH levels, whereas intracellular GSH contents were unaffected. Plasma concentrations of oxidized glutathione (GSSG) increased up to 5-fold in GSH-treated animals suggesting counteraction of the vascular oxidant stress produced by activated KC. CONCLUSIONS: Intravenous GSH administration during reperfusion of ischemic livers prevents reperfusion injury in rats. Because GSH is well tolerable also in man, this novel approach could be introduced to human liver surgery.

Kinin-B1 receptors in ischaemia-induced pancreatitis: functional importance and cellular localisation.

In this study we compare the role of kinin-B1 and B2 receptors during ischaemia/reperfusion of rat pancreas. Our investigations were prompted by the observation that infusion of a kinin-B2 receptor antagonist produced significant improvement in acute experimental pancreatitis. In an acute model with two hours of ischaemia/two hours of reperfusion, application of the kinin-B1 receptor antagonist (CP-0298) alone, or in combination with kinin-B2 receptor antagonist (CP-0597), significantly reduced the number of adherent leukocytes in post-capillary venules. In a chronic model with five days of reperfusion, the continuous application of kinin-B1 receptor antagonist or a combination of kinin-B1 and B2 receptor antagonists markedly reduced the survival rate. In kinin-receptor binding studies kinin-B1 receptor showed a 22-fold increase in expression during the time of ischaemia/reperfusion. Carboxypeptidase M activity was up-regulated 10-fold following two hours of ischaemia and two hours of reperfusion, provided the appropriate specific ligand, des-Arg10-kallidin and/or des-Arg9-bradykinin, was used. The occurrence of kinin-B1 receptor binding sites on acinar cell membranes was demonstrated by micro-autoradiography. With a specific antibody, the localisation of kinin-B1 receptor protein was confirmed at the same sites. In conclusion, we have demonstrated the up-regulation of the pancreatic acinar cell kinin-B1 receptors during ischaemia/reperfusion. The novel functional finding was that antagonism of the kinin-B1 receptors decreased the survival rate in an experimental model of pancreatitis.

Influence of platelet-derived growth factor on microcirculation during normal and impaired wound healing.

The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.