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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

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.

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.

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.

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.

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.

Components of the activated complement cascade are considered to play a pivotal role in ischemia-reperfusion-induced organ injury. With the use of intravital epifluorescence microscopy, we investigated the effect of complement inhibition by the recombinant soluble complement receptor 1 (sCR1; TP10) on the effect of macromolecular microvascular permeability, functional capillary perfusion, and leukocyte endothelium interaction in postischemic pancreatitis. Anaesthetized Sprague-Dawley rats were subjected to 60 min of normothermic pancreatic ischemia induced by microclipping of the blood-supplying arteries of the organ. Rats who received sCR1 (15 mg/kg body wt iv; n = 7) during reperfusion showed a significant reduction of permeability (1.77 ± 1.34 × 10-8cm/s; n = 7) of tetramethylrhodamine isothiocyanate-labeled albumin injected 90 min after the onset of reperfusion compared with vehicletreated animals (6.95 ± 1.56 × 10-8cm/s; n = 7). At 120 min after the onset of reperfusion, the length of red blood cell-perfused capillaries (functional capillary density) was significantly improved (from 279 ± 15.7 to 330 ± 3.7 cm-1; n = 7) and the number of leukocytes adherent to postcapillary venules was significantly reduced (from 314 ± 87 to 163 ± 71 mm-2; n = 7) by sCR1 compared with vehicle treatment. Complement inhibition by sCR1 effectively ameliorates pancreatic ischemia-reperfusion-induced microcirculatory disturbances and might be considered for treatment of postischemic pancreatitis.

Temporary clamping of the portal triad, ie, inflow occlusion by the Pringle maneuver,1 is a common strategy to minimize bleeding during hepatic resection. However, portal triad clamping can induce relevant ischemia reperfusion injury (IRI) and failure of the remnant liver.2–7 Because prolonged ischemia is frequently unavoidable to achieve radical tumor resection,2 therapeutic strategies to minimize IRI are needed. The precise sequence of events leading to IRI following warm liver ischemia has not been completely elucidated. However, there is substantial evidence that activation of Kupffer cells (KC), the generation of reactive oxygen species, (ROS) and disturbances of the hepatic microcirculation contribute to reperfusion injury.6,8–12 During reperfusion activated KC produce mediators of inflammation and release ROS into the sinusoidal space.6,12 The resulting vascular oxidant stress has been discovered as potential mechanism of hepatic perfusion failure.13–16 ROS can activate redox-sensitive transcription factors, thereby activating proinflammatory genes and adding to the hepatic damage.17,18 Furthermore, ROS may contribute to the activation of calcium–dependent proteases (calpains)19 which mediate several intracellular processes resulting in necrotic or apoptotic cell death.20,21 Thus, ROS can be considered as signal molecules, which trigger several pivotal mechanisms of reperfusion injury. Consequently, antioxidant strategies appear a promising approach to prevent hepatic reperfusion injury.10 Recent studies investigated the therapeutic potential of the endogenous antioxidant glutathione (GSH), in particular since it has a low toxicity in humans22 and is cost-effective. Earlier studies indicated that GSH is able to react spontaneously with nearly all oxidants formed during inflammation.23–25 Subsequent studies demonstrated that GSH released through the GSH transporter of hepatocytes may act as an endogenous defense system against KC–derived ROS, thereby protecting the hepatic vasculature from damage by KC.8 The hepatic GSH content which can decrease rapidly during preoperative starvation is pivotal for this defense system.26 Thus, any intervention increasing hepatic or plasma GSH levels should convey protection against reperfusion injury. Compounds with potential therapeutic value include N–acetylcysteine,27 methionine,28 glutathione esters29 but also GSH itself. As shown recently in vitro, treatment of cold preserved livers with 2 or 4 mM GSH upon reperfusion prevented cell damage to hepatocytes in the model of cell-free rat liver perfusion.30 Therefore, the purpose of this study was to test the hypothesis that intravenous administration of GSH protects the rat liver against reperfusion injury after prolonged warm ischemia in vivo. In particular, the aims of the current study were: to investigate a potential influence of intravenously applied GSH on necrotic and apoptotic cell death, disturbances of the hepatic microcirculation and animal survival and to determine the functional significance of changes of the extra- and intracellular antioxidant capacity.

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 N2O/O2 mixture and saturated with 32-36% FiO2. During observations the body temperature was kept constant at 37°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, paO2 and pCO2 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.

Measurement of regional organ blood flow by means of fluorescent microspheres (FM) is an accepted method. However, determination of regional portal blood flow (RPBF) cannot be performed by microspheres owing to the entrapment of the spheres in the upstream capillary bed of the splanchnic organs. We hypothesized that an adequate experimental setting would enable us to measure RPBF by means of FM and to analyze its distribution within the pig liver. A mixing chamber for the injection of FM was developed, and its capability to distribute FM homogeneously in the blood was evaluated in vitro. The chamber was implanted into the portal vein of six anesthetized pigs (23.5 ± 2.9 kg body wt). Three consecutive, simultaneous injections of FM of two different colors into the chamber were performed. Reference portal blood samples were collected by means of a Harvard pump. At the end of the experiment, the liver was explanted and fixed in formalin before dissection. FM were isolated from the tissue samples by an automated process, and fluorescence intensity was determined. Comparison of 5,458 single RPBF values, determined by simultaneously injected FM, revealed good agreement (bias 2.5%, precision 12.7%) and high correlation ( r = 0.97, r2 = 0,95, slope = 1.04, intercept = 0.05). Median RPBF was 1.07 ± 0.78 ml · min-1 · g-1. Allocation of the blood flow values to the anatomic regions of the liver revealed a significantly higher RPBF ( P = 0.01) in the liver tissue located close to the diaphragm compared with the rest of the organ and a significantly lower RPBF ( P = 0.01) in the left liver lobe compared with the median and right lobes. The results show that the model presented makes it possible to measure RPBF by means of FM reliably and that RPBF is distributed heterogeneously in the porcine liver.

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.

OBJECTIVE Changes of major cerebral vessels in patients with subarachnoid hemorrhage (SAH) are well known from routine cerebral angiography. Data on changes in the microcirculation do not exist. This study sought to provide a qualitative and quantitative analysis of the cortical microcirculation after SAH. METHODS By means of orthogonal polarization spectral imaging, a qualitative and quantitative analysis of cortical microcirculation was performed during aneurysm surgery in 3 patients with an incidental intracerebral aneurysm and 10 patients with SAH. Vessel diameters, red blood cell velocity, and functional capillary density were analyzed before and after the aneurysm was clipped. RESULTS Initial capillary density in patients with an incidental aneurysm was 91.5 +/- 36.5 cm(-1) (mean +/- standard deviation) compared with 30.5 +/- 13.8 in patients with SAH (P < 0.05). In patients with SAH, capillary density increased significantly to 53.9 +/- 29.1 cm(-1) (P < 0.05) during the operation, as did the frequency of venules with a red blood cell velocity greater than 2 mm/s (P < 0.05). No significant change of arteriolar or venular diameters was observed. However, in patients with SAH, mono- and multisegmental microvasospasms in arterioles were observed, with a reduction of vessel diameters up to 75.1%. CONCLUSION Orthogonal polarization spectral imaging is a suitable method to study cerebral microcirculation during surgery. In patients with SAH, capillary density is significantly decreased and small arteries and arterioles of the cortical surface exhibit vasospasm that cannot be detected by angiography or transcranial Doppler sonography. These changes may contribute to the initial clinical symptoms and may have an influence on the clinical postoperative course.

Activated platelets release angiogenic growth factors and have therefore been proposed to contribute to tumor angiogenesis within a potentially prothrombotic tumor microcirculation. The aim of the study was to investigate interactions of platelets with the angiogenic microvascular endothelium of highly vascularized solid tumors during growth and in response to endothelial stimulation in comparison with normal subcutaneous tissue. Experiments were performed in the dorsal skinfold chamber preparation of C57BL/6J mice bearing the Lewis lung carcinoma (LLC-1) or methylcholanthrene-induced fibrosarcoma (BFS-1). Fluorescently labeled rolling and adherent platelets, red blood cell velocity, and vessel diameters were assessed by intravital fluorescence microscopy on days 1, 3, 8, and 14 after tumor cell implantation. Slightly elevated numbers of rolling platelets were observed in the early stages of tumor angiogenesis at day 1 (control, 1.7 ± 0.6; LLC-1, 3.4 ± 1.8; BFS-1, 3.0 ± 0.7 [1/mm/s],P

Introduction: Capillary perfusion failure of the pancreatic microcirculation is characteristic in the pathogenesis of acute pancreatitis and ischemia-reperfusion damage after pancreas transplantation. Up to now, no logistic suitable method for analyzing pancreatic capillary perfusion during operations in humans has been established without the use of fluorescent dyes. Aim: To compare the well-established technique of intravital epifluorescence microscopy with the novel noninvasive method of orthogonal polarization spectral (OPS) imaging for measurement of the pancreatic functional capillary density. Methodology: In eight anesthetized rats, six identical capillary regions of interest per animal were measured by both methods, and the results were compared. Results: Absolute values from the capillary perfusion data were not significantly different between the two methods (fluorescence microscopy: 394 ± 44 cm/cm 2 ; OPS imaging: 385 ± 45 cm/cm 2 ). Correlation parameters were significant, and Bland-Altman analyses showed good agreement with a mean difference (bias) between the two methods of 6.9 cm/cm 2 , indicating that slightly smaller values are measured with OPS imaging. Conclusion: OPS imaging is a valid noninvasive method that analyzes the pancreatic microcirculation as accurately as the established intravital microscopy technique and therefore could be useful for clinical research and diagnosis during transplantation and operations.

Platelets are suggested to participate in the pathogenesis of hepatic ischemia-reperfusion (I/R) injury. This study was designed to analyze platelet-endothelial cell interactions in the postischemic mouse liver in vivo and to define the role of endothelial versus platelet P-selectin for these interactions. Platelet-endothelial cell interactions were quantitatively analyzed using intravital fluorescence microscopy after lobar hepatic I/R in C57BL/6 wild-type and P-selectin-deficient mice after infusion of ex vivo rhodamine-6G-labeled wild-type and P-selectin-deficient platelets. Reperfusion injury and apoptosis were assessed by established methods. In wild-type animals, hepatic I/R caused significantly enhanced platelet-endothelial cell interactions in terminal arterioles and postsinusoidal venules as well as platelet stagnation in sinusoids. Concomitantly, transaminase and caspase-3 activities were elevated and sinusoidal perfusion was impaired. In contrast, platelet-endothelial cell interactions were nearly absent in arterioles and venules of mice lacking endothelial P-selectin, irrespective of the presence of P-selectin on infused platelets, but still significantly elevated in sinusoids. Simultaneously, sinusoidal perfusion failure was ameliorated, and transaminase- and caspase-3 activities were significantly reduced in P-selectin-deficient mice as compared with wild-type animals. The present intravital microscopic study provides, for the first time, quantitative analyses of platelet-endothelial cell interactions in the postischemic hepatic microcirculation. Our in vivo data show that endothelial P-selectin is critical for postischemic platelet-endothelial cell interactions within hepatic presinusoidal arterioles and postsinusoidal venules. P-selectin deficiency prevents microvascular injury and apoptosis after warm hepatic I/R.

Introduction: Microcirculatory disturbances caused by ischemia-reperfusion injury (IRI) are the crucial hallmarks of pancreatitis following pancreas transplantation. Aims: To develop a novel rodent model of normothermic in situ ischemia of a pancreatic tail-segment that simulates the clinical situation of pancreas transplantation by flushing the organ via an inserted microcatheter and thus enables selective treatment of the organ via this access. Methodology: Four experimental groups were investigated (n = 7 Wistar rats/group): sham animals without ischemia and dissection of the pancreas; control animals with dissection of a pancreatic tail segment pedunculated on the splenic vessels and flushing od this segment with saline via a microcatheter; and two groups of animals treated like controls with a pancreatic ischemia time of 1 hour or 2 hours. With use of intravital epifluorescence microscopy, the microcirculatory damage was characterized by investigation of functional capillary density (FCD) and leukocyte adherence in postcapillary venules (LAV) before ischemia and during a reperfusion time of 2 hours. Dry:wet ratio determinations, light microscopy, and electron microscopic investigations were performed to characterize the histologic organ damage. Results: FCD decreased significantly (p < 0.05) 2 hours after reperfusion in the groups of 1-hour (-29.21%) and 2-hour ischemia (-42.73%), in comparison with baseline values. LAV increased significantly (p < 0.05), 4.3- and 5.8-fold, after 1-hour and 2-hour ischemia during the observation time. The histologic damage was similar to post-transplantation pancreatitis in humans 1 hour after reperfusion. In sham and control animals these alterations were not significant. Conclusions: The rodent in situ model of pancreatic IRI showed standardized microcirculatory damage dependent on the ischemia time. Offering the possibility of selective treatment by the direct artery access to the ischemic pancreatic area, the model enables investigations of questions related to human pancreas transplantation.

Intravital fluorescence microscopy (IVM) is a widely used method to study the microcirculation in several organs. Our aim was to develop a standard rat model to evaluate the microcirculatory characteristics of the urinary bladder under physiological pressure conditions using the most advanced fluorescence videomicroscopic techniques. Spraque-Dawley rats were used after filling their bladders with a constant volume of saline solution. The intravesical pressure was continuously monitored. The bladder was positioned on a specially designed stage and IVM measurements were made at the beginning, the 90th, 120th and 180th min. Arteriolar and venular diameters, functional capillary density, venular red blood cell velocity, arteriolar and venular macromolecular leakage and leukocyte-endothelial cell interactions (observation of rolling and firmly adherent leukocytes) were quantitatively assessed by a computer assisted analysis system. Neither microcirculatory parameters nor the intravesical pressure changed significantly during the observation period of 180 min using constant filling volume. We successfully established a new, well functioning and reproducible model to study the microcirculation of the rat bladder using intravital fluorescent microscopy.

BACKGROUND Microcirculatory failure, activation of Kupffer cells (KC), and the formation of reactive oxygen species (ROS) are considered pivotal mechanisms of reperfusion injury after orthotopic liver transplantation. However, the sequence of these events and their impact on early graft function remain controversial. We therefore investigated whether KC induce microcirculatory disturbances through ROS release and whether microcirculatory failure contributes to early graft function after liver transplantation. METHODS Donor livers of Lewis rats were pretreated either with saline or with gadolinium chloride (GdCl3), an inhibitor of KC function (n=8 each). Syngeneic OLT was performed after 24 hr of hypothermic preservation in University of Wisconsin solution. RESULTS Intravital microscopy revealed significantly higher sinusoidal perfusion rates in GdCl3-treated allografts (92+/-1.1% vs. 75.7+/-0.8%; P

Background Although hepatic ischemia-reperfusion (I/R) injury can be reduced by cooling of the ischemic organ, a systematic in vivo analysis of the influence of organ temperature in I/R injury is missing. The aim of this study was to systematically investigate the impact of defined temperatures of the ischemic liver tissue on microvascular I/R injury. Methods Ischemia of the left liver lobe was induced in C57BL/6 mice for 90 min. The ischemic lobe was placed in a polyethylene well and the temperature was adjusted to 37 degrees C, 26 degrees C, 15 degrees C, and 4 degrees C by superfusion with cooled/warmed saline solution. The ischemia groups (n=7 each) were compared with a sham-operated group (n=7). The sinusoidal perfusion index and the number of leukocytes firmly adherent to the endothelium of postsinusoidal venules were assessed using intravital fluorescence microscopy at 30 min, 120 min, and 240 min of reperfusion, respectively. At the end of the experiment, serum activities of the liver enzymes aspartate aminotransferase/alanine aminotransferase were determined, and tissue specimens were examined by electron microscopy. Results Core body temperature did not differ significantly between the groups. In the 37 degrees C group, the sinusoidal perfusion index was significantly reduced and the number of adherent leukocytes was significantly increased compared with the sham group. In all hypothermia groups, however, the microcirculatory parameters did not differ from the sham group. Serum activities of aspartate aminotransferase/alanine aminotransferase were significantly increased and hepatocellular integrity was severely affected in the 37 degrees C group as compared with all other groups. Conclusions These findings demonstrate that in the mouse liver the known protective effect of hypothermia is already encountered at 26 degrees C. Further reduction of temperature did not generate additional protection from I/R injury.

BACKGROUND: The aim of this study was to investigate changes in the blood microcirculation of skin, subcutaneous tissue, and striated muscle, and the venous and lymphatic outflow from hind limb, after a standardized mechanical trauma. METHODS: Trauma, defined as 50% of the minimal energy needed for tibia fracture (3.7 J/g), was applied to the leg of hairless mice. Intravenously injected fluorescein isothiocyanate-dextran 150 kDa and Rhodamine-6G were used for intra-vital fluorescence microscopy of blood vessels. Lymphatics were stained with fluorescein isothiocyanate-dextran injected into the footpad. A computer-assisted analysis system allowed measurement of the functional capillary density (FCD), vessel diameters, velocity of blood flow, and edema value expressed as extravasation index (EV). The percentage of slowly rolling and sticking leukocytes in postcapillary venules was estimated. RESULTS: At the site of injury, trauma resulted in significant reduction of FCD in skin, subcutaneous tissue, and striated muscle. There were no significant differences in the vessel diameter (skin subcutaneous and muscle arterioles and venules, and superficial saphenous artery and vein) or velocity of blood flow (subcutaneous tissue and muscle venules). The EV increased significantly in muscle venules and was higher in muscles, subcutaneous tissue, and superficial saphenous veins than in controls (nonsignificantly). An increased percentage of slowly rolling and sticking leukocytes was noted in the superficial saphenous vein at the site of injury and proximal to it. The lymphatics remained patent, with faster visualization and increased summarized cross-sectional areas in traumatized extremities. CONCLUSION: Early changes occurring in soft tissues in response to mechanical injury were characterized by reduction in FCD of skin and muscles, and less in subcutis; increased EV, reflecting leakage of macromolecules; increased percentage of slowly rolling and sticking leukocytes; maintenance of lymphatic vessel continuity; and increased lymph formation and flow rate.

Background: Gastric tonometry is intended to reveal alterations in splanchnic perfusion and oxygenation. Based on the tonometric measurement of gastric mucosal partial pressure of carbon dioxide (pCO2) and the simultaneous determination of arterial blood gas parameters (bicarbonate concentration [HCO3–], pH and pCO2), several parameters can be calculated. Aims: To identify the most suitable tonometric parameter [gastric mucosal pH (pHi), intramucosal pCO2 (piCO2), the difference between tonometric and arterial pCO2 concentrations (pCO2 gap), [H+] gap] that reliably reflects gastric hypoperfusion and hypoxia during severe haemorrhagic shock. Design: Randomised, controlled experimental study. Methods: An artificial stenosis of the left anterior descending coronary artery (LAD) was induced. Subsequently, the animals were haemorrhaged to a mean arterial pressure of 45 mmHg, which was maintained for 60 min. Measurements and main results: Tonometric measurements were performed in 17 land-race pigs before and after induction of LAD stenosis and after haemorrhagic shock. P values obtained using the Wilcoxon signed-rank testing were used to compare the level of significance for the tonometric parameters and the corresponding arterial blood gas values [arterial pCO2 (paCO2), [HCO3–], arterial pH (pHa)]. While induction of critical coronary stenosis did not provoke any changes, all parameters changed significantly during haemorrhagic shock. The lowest P value was found for pHi (P=0.00013) followed by [H+ gap] (P=0.0005). P values higher by a factor of ten were found for pCO2 gap (P=0.00119) and were highest for piCO2 (P=0.00562). P values of the corresponding arterial blood gas parameters were lower by a factor of ten than the P value of piCO2. Conclusion: pHi, pCO2 gap and [H+] gap are considerably influenced by changes of systemic arterial blood gas values. This is demonstrated by lower P values of the corresponding arterial blood gas values in comparison with piCO2. Therefore pHi, pCO2 gap and [H+] gap seem to indicate more likely systemic changes, whereas piCO2 appears to reflect disturbances of regional gastric tissue perfusion and oxygenation more reliably than any other derived tonometric parameter.

Extracorporeal shock-wave application facilitates dissolution of rotator cuff calcifications. Therefore, disappearance or disintegration of tendon calcifications by shock waves might be appropriate for any kind of tendon calcification. Here, shock waves with various energy flux densities were applied to the mineralized medial gastrocnemius tendon of turkeys as an animal model. After application of shock waves in vivo, with energy flux density of 0.6 mJ/mm(2), histologic examination and microradiography did not show dissolution or disintegration of tendon calcifications. After shock-wave application in vitro, even for energy flux density of 1.2 mJ/mm(2) neither dissolution nor disintegration of tendon calcifications were observed. Biomechanical testing revealed significant impairment of tensile strength following shock-wave application in vitro, with energy flux density of 1.2 mJ/mm(2), but not with 0.6 mJ/mm(2). These results are important for considerations of clinical extracorporeal shock-wave application on tendon calcifications, as well as on tendon ossifications.

Tissue macrophages (M phi) play a central and essential role in modulating the initiation and perpetuation of the inflammatory response. Phenotypical and functional differences among alveolar M phi (AM) and peritoneal M phi (PM) have been reported, but less is known about pleural M phi (PLM) and their ability and capacity to release biologically active substances. Therefore, the aim of this study was to determine the production of superoxide anion, nitric oxide (NO), and tumor necrosis factor alpha (TNF-alpha) by PLM in comparison to AM and PM in vitro. M phi from rats were isolated by lavage of the respective body compartment and characterized by evaluating the expression of the surface antigens MHC class II molecules, CD11b, and ED2-like antigen. Upon activation, AM produced significantly higher amounts of superoxide anion, NO, and TNF-alpha compared to PM and PLM. Taken together, the findings of this study demonstrate that rat PLM resemble PM more than AM in terms of production of key inflammatory mediators.

Background: A newly developed observation chamber has been designed for comfortable limb immobilization during intravital microscopic analysis, which permits direct, repeated, long-lasting observations of the microcirculation in the various hindleg soft tissues. Methods: Experiments were performed under inhalation isoflurane/nitrous oxide anesthesia. Intravenously injected fluorescein isothiocyanate (FITC)-dextran (M r 150,000) and Rhodamine 6G (Sigma, St. Louis, MO) allowed for visualization of both microcirculatory phenomena in arterioles, capillaries, and venules and macrocirculatory structures as superficial saphenous artery and vein. Skin microcirculation analysis was performed from the epidermal side (group A, n = 7), whereas observation of deeper situated tissues was performed after oval skin excision on the medial surface of the tibial area (group B, n = 7). FITC-dextran (M r 150,000; group C, n = 8) injected into the foot pad permitted visualization of venous, arterial, and accompanying lymphatic vessels. With the aid of a computer-assisted microcirculation analysis system, functional capillary density, vessel diameter, edema formation, and leukocyte-endothelial cell interactions were evaluated. The ratio of rolling leukocytes, given as percentage of all leukocytes passing the vessel segment during a 30-second observation interval, and the number of sticking leukocytes per square millimeter of endothelial surface were determined. Results: This new model allows the analysis of the complex in vivo changes of macro- and microcirculatory parameters in the different (venous, arterial, lymphatic) vessels of the covering tissues (skin and muscle) of the mouse hindleg. Conclusion: The potential applications of this technique include the study of mechanical trauma, ischemia-reperfusion injury, and tissue compression mimicking both acute and prolonged venous stasis on both the microcirculatory and macrocirculatory levels in the different tissue compartments.

Hypotension in brain-dead organ donors is considered a determinant factor of graft viability. The aim of this study was to elucidate the role of hypotension in brain-death associated impairment of hepatic microcirculation and function. Male Sprague-Dawley rats with an intracranial balloon were used. Group I (n = 7) served as sham controls. In group II (n = 7) brain death was induced through inflation of an intracranial balloon. In group III (n = 7) hypotension without brain death was induced by means of pentobarbital. In group II, a steep rise of arterial pressure was followed by a fall to a lower level (P < 0.01, vs. group I). Also in group III arterial pressure was lower (P < 0.01, vs. group I). In group II, bile production was diminished (P < 0.05). Impaired sinusoidal perfusion (P < 0.01) and enhanced leukocyte endothelium interaction (P < 0.05) were documented in hepatic microvasculature. Electron microscopic analysis revealed vacuolization of hepatocytes; these changes were not observed in group III. Brain death induces specific changes of liver microcirculation, function and histomorphology. Independent of associated hypotension, brain death per se impairs donor liver graft quality.

Background. Experimental studies demonstrated a suppression of oxygen-derived free radicals, reduced adhesion of activated neutrophils on the endothelium and an increase of de novo synthesis of surfactant during liquid ventilation with perflurocarbon. The purpose of this study was to assess the pulmonary graft function after preservation with endobronchially administered perfluorocarbon as an alternative to flush perfusion. Methods. Native bred pigs underwent orthotopic left lung transplantation. Donor lungs were flushed in situ with either a low-potassium dextran solution (LPD, n=6) or a perfluorochemical was administered endobronchially (PFC, n=6) and were then stored after removal for 18 hr at 4°C. Pulmonary graft function was assessed after reperfusion for 5 hr by measuring pulmonary gas exchange and hemodynamics during isolated ventilation and perfusion. Tissue specimens were taken for analysis of morphology and wet/dry ratio. All values were compared to a sham-operated group (n=6). Results. Pulmonary gas exchange of the graft revealed reduced paO 2 values and elevated paCO 2 values in the PFC group throughout the observation period as compared with the LPD group and sham group. Endothelial alterations and fibrin exudate in the PFC group were significantly more pronounced. Lungs in the LPD group showed functional and morphological recovery close to sham group. Conclusions. Long-term preservation with endobronchially administered perfuorocarbon is possible. Impaired pulmonary graft function and pronounced morphological alterations indicate an aggravation of the ischemic reperfusion injury after lung transplantation compared to LPD preserved lungs.

Background Perfluorocarbon-based oxygen carriers have been proposed as an adjunct to autologous blood conservation techniques during elective surgery. To date, the effects of perfluorocarbon emulsions at the microcirculatory level have not been studied extensively. In this study the effects of perflubron emulsion on the microcirculation after acute normovolemic hemodilution (ANH) were investigated using different colloid plasma expanders. Methods The dorsal skin fold chamber model and intravital fluorescence microscopy were used for analysis of the microcirculation in the thin striated skin muscle of conscious hamsters (body weight, 40-60 g). Measurements of microvascular perfusion and leukocyte adhesion (n = 6 animals per experimental group) were made before and at 10, 30, and 60 min after ANH (to hematocrit 0.3) with either 6% hydroxyethyl starch 200/0.6 (HES), 3.5% gelatin, 5% human serum albumin (HSA), or 6% dextran 60 (DX-60) followed by intravenous injection of 3 ml/kg body weight of a 60% weight/volume perfluorocarbon emulsion based on perflubron (perfluorooctyl bromide) emulsified with egg yolk lecithin. Results Acute normovolemic hemodilution with HES, gelatin, or HSA followed by injection of perflubron emulsion elicited no alterations of local microvascular perfusion or leukocyte-endothelium interaction as assessed in arterioles and postcapillary venules. However, ANH with DX-60 followed by injection of perflubron emulsion led to a significant reduction of erythrocyte velocity in postcapillary venules and an increase in venular leukocyte sticking that was never observed with DX-60 alone. Conclusions Hydroxyethyl starch, gelatin, and HSA are compatible with perflubron emulsion in the setting of ANH. Only DX-60 appeared to be incompatible with perflubron emulsion, as evidenced by impairment of capillary perfusion.

Intravital microscopy is widely used to study the microcirculation. However, the use of fluorescent dyes can induce phototoxic effects which may affect the measurements, particularly in tissue exposed to oxidative stress. The aim of the study was to determine the threshold light dose at which fluorescent microscopy is associated with phototoxic effects in the hamster dorsal skinfold chamber under normal and pathological conditions. The extent of phototoxicity in the microcirculation in the hamster skinfold chamber was investigated using intravital fluorescent microscopy during 60 min of illumination (1048 mW/cm2) applying two different concentrations of fluorescein isothiocyanate dextran under baseline conditions (groups A and B) and following 4 h of ischemia (groups C and D). In the second part of the study the microvasculature was analyzed regarding phototoxic effects during a standardized intravital microscopic examination after 4 h of pressure induced ischemia. Groups I and II (n=7) were studied using epiillumination after injection of fluorescein isothiocyanate dextran plus rhodamine 6G or rhodamine 6G only. In group III (n=7) only transillumination was used. Arteriolar vasospasm, microvascular perfusion failure, thrombus formation, and enhanced leukocyte endothelium interaction were observed as signs of a phototoxic effect in normal tissue. However, the light doses needed to induce these effects clearly exceeded those during standard examinations. The induction of a 4-h ischemia and reperfusion further enhanced these effects. Despite the predamage by ischemia/reperfusion the comparison of epiillumination and transillumination microscopy using a standard protocol showed no differences regarding the parameters analyzed at any time. This indicates that epiillumination and the fluorescent dyes per se did not affect the experimental results. These results show that ischemia/reperfusion studies in the dorsal skinfold chamber of the Syrian golden hamster can be carried out safely without the risk of inducing phototoxic effects by fluorescent microscopy. Nevertheless every laboratory using epiillumination and fluorescent dyes should take precautions to avoid these effects by the use of sensitive cameras to lower the light dose.

Sublethal hyperthermia and the following recovery from this heat exposure, referred to as hyperthermic preconditioning, elicits a transient state of tolerance to oxidative insults through an intracellular protective response: stress response. The impact of hyperthermic preconditioning on hepatic microcirculatory disturbance, which is one of the determinants of ischemia/reperfusion-induced injury of the liver, was investigated by using intravital fluorescence microscopy. Thirty minutes of ischemia and a subsequent 120 minutes of reperfusion was induced in an in situ isolated perfusion model of Sprague-Dawley rats. Heat stress was given by whole-body hyperthermia, and a subsequent recovery was allowed for 18 or 48 hours, respectively. Postischemic decrease in sinusoidal perfusion rate and sinusoidal diameter, leukocyte stagnation in sinusoids, and leukocyte adhesion in postsinusoidal venules were significantly attenuated in both hyperthermia-pretreated groups. A recovery of bile production, a reduction of liver enzyme release, and an attenuation of tissue edema and histological damage were also observed. A marked expression of heat shock protein (HSP) 70 and heme oxygenase (HO-1)/HSP32 was correlatively observed in the liver tissue coincident with the induction of these protective effects. Hyperthermic preconditioning provides a continuous long-term and constant inhibitory effect (up to 48 hours after heat exposure) on postischemic injury of the liver through the attenuation of microcirculatory disturbances. These beneficial effects might be associated with a concomitant increase in HSP70 and HO-1/HSP32 expression.

Hemorrhagic shock alters heterogeneity of regional myocardial perfusion (RMP) in the presence of critical coronary stenosis in pigs. Conventional resuscitation has failed to reverse these effects. We hypothesized that improvement of the resuscitation regime would lead to restoration of RMP heterogeneity. Diaspirin-cross-linked hemoglobin (10 g/dl; DCLHb) and human serum albumin (8.0 g/dl; HSA) were used. After baseline, a branch of the left coronary artery was stenosed; thereafter, hemorrhagic shock was induced. Resuscitation was performed with either DCLHb or HSA. At baseline, the fractcal dimension ( D) of subendocardial myocardium was 1.31 ± 0.083 (HSA) and 1.35 ± 0.106 (DCLHb) (mean ± SD). Coronary stenosis increased subendocardial D slightly but consistently only in the DCLHb group (1.39 ± 0.104; P < 0.05). Shock reduced subendocardial D: 1.21 ± 0.093 (HSA; P = 0.10), 1.25 ± 0.092 (DCLHb; P < 0.05). Administration of DCLHb increased subendocardial D in 7 of 10 animals (1.31 ± 0.097; P = 0.066). HSA was ineffective in this respect. DCLHb infusion restored arterial pressure and increased cardiac index (CI) to 80% of baseline values. Administration of HSA left animals hypotensive (69 mmHg) and increased CI to 122% of the average baseline value. Shock-induced disturbances of the distribution of RMP were improved by administration of DCLHb but not by HSA.

The CytoscanTM Model E-II (Cytometrics Inc., Philadelphia, Pa., USA) is a newly developed instrument which functions as an intravital microscope and is small and easily portable. Through the use of orthogonal polarization spectral (OPS) imaging, the Cytoscan Model E-II delivers images of the microcirculation which are comparable to those achieved with intravital fluorescence videomicroscopy (IFM), but without the use of fluorescent dyes. The purpose of this study was to validate the Cytoscan Model E-II instrument against IFM. The experiments were carried out on striated muscle in the dorsal skinfold chamber of the awake Syrian hamster. The following parameters were measured in identical regions of interest in the same animal under baseline conditions and 0.5 and 2 h after a 4-hour period of pressure-induced ischemia: arteriolar diameter, venular diameter and venular red blood cell velocity. Bland-Altman plots showed good agreement between the two techniques for venular red blood cell velocity. As expected, arteriolar and venular diameters as measured by the Cytoscan were on average 5 µm smaller than the values from IFM, since the Cytoscan measures the red blood cell column width and IFM measures luminal diameter. Thus, OPS imaging can be used to make valid measurements of microvascular diameter and red blood cell velocity in tissues.