A novel approach for comparative study of periosteum, muscle, subcutis, and skin microcirculation by intravital fluorescence microscopy

Herein, we report a new model, which allows comparative study of the microcirculation of different peripheral tissues, i.e., periosteum, skeletal muscle, subcutis, and skin. Using dextran-insensitive Wistar rats gracilis and semitendinosus muscles of the left hindlimb were prepared in association with their appertaining tibial fragments, subcutis, and skin. Blood supply was guaranteed by the femoral artery via the saphenous vessels. High-resolution intravital epi-illumination microscopy of the two muscles displayed the typical microvascular architecture with the capillaries running in parallel to each other (capillary density (CD) 128.4 +/- 4.5 cm-1). In subcutis and skin, capillaries were found arranged as interconnecting mesh-like networks with a density, which was significantly higher (P < 0.05) in subcutis (191.0 +/- 5.5 cm-1) compared with skin (108.9 +/- 3.3 cm-1). Analysis of periosteal tissue revealed two distinct types of arrangements of microvascular architecture. Adjacent to the major feeding and draining vessels of the periosteum, capillaries were organized in densely meshed shunt-like networks, revealing the highest capillary density (242.7 +/- 13.2 cm-1; P < 0.05) of all tissues studied. Periosteal capillaries distant from the major feeding and draining vessels were arranged in parallel to the longitudinal axis of the tibial bone and presented with a density similar to that of the skeletal muscle (128. 6 +/- 9.4 cm-1). Topical application of acetylcholine for analysis of physiological reactivity of the microvasculature showed dose-dependent arteriolar dilation. Moreover, a 3-min upstream femoral artery occlusion demonstrated an appropriate hyperemic response in all tissues studied, indicating intact myogenic control. A prolonged period of ischemia (120 min) followed by reperfusion (60 min) caused massive (P < 0.05) leukocyte-endothelial cell interaction in postcapillary venules, similarly as reported in other microvascular tissue preparations. We propose that the model presented provides a good approach to all peripheral tissues for both the analysis of the physiology of tissue-confined microvascular control and the development of novel therapeutic strategies to counteract manifestation of nutritional dysfunction and inflammatory response in disease.     

Subchronic dietary toxicity of strychnine: bobwhite quail are less sensitive than mallard ducks

PURPOSE: This study was undertaken to evaluate in vivo the effect of recombinant hirudin (r-hirudin [HBW 023]), a potent thrombin inhibitor, on the process of microvascular thrombus formation and recanalization. METHODS: Thrombosis was induced photochemically in distinct arterioles (n = 25) and venules (n = 30) of the ear of 16 hairless hr/hr mice (8 to 10 weeks old, 25 to 30 g of body weight). r-Hirudin (1 mg/kg of body weight) was administered intravenously directly before thrombus induction; saline-treated animals served as controls. Thrombus formation (i.e., first platelet deposition at the endothelial lining [FPD]; inner luminal diameter reduction to 50% [D/2]; complete vessel occlusion [CVO]), vessel recanalization, microcirculatory parameters, and leukocyte-endothelial cell interaction were analyzed by means of intravital fluorescence microscopy. RESULTS: Hirudin significantly delayed the process of thrombus formation compared with saline-treated controls in both arterioles (FPD: 381 +/- 80 vs 137 +/- 25 seconds, P < 0.05; D/2: 627 +/- 49 vs 501 +/- 71 seconds; CVO: 925 +/- 78 vs 854 +/- 60 seconds) and venules (FPD: 173 +/- 11 vs 59 +/- 4 seconds; D/2: 342 +/- 54 vs 228 +/- 27 seconds; CVO: 541 +/- 85 vs 344 +/- 43 seconds; P < 0.05). In addition, r-hirudin-treated animals showed an increased rate of vessel recanalization at 24 hours after thrombus induction (arterioles: 54% [7 of 13] vs 0% [0 of 12], P < 0.05; venules: 77% [10 of 13] vs 53% [9 of 17]), whereas microcirculatory parameters and leukocyte-endothelial cell interaction were not affected. CONCLUSION: Our data indicate that r-hirudin not only counteracts the process of thrombus formation but also promotes vessel recanalization, thus supporting its use in clinical microvascular surgery.     

Effects of Carolina rinse and adenosine rinse on microvascular perfusion and intrahepatic leukocyte-endothelium interaction after liver transplantation in the rat

Flushing hepatic grafts immediately before revascularization with a specially designed rinse solution such as "Carolina rinse" has been reported to improve survival after liver transplantation in the rat. This study investigated the influence of Carolina rinse and adenosine rinse on early graft function, microcirculation, and leukocyte (WBC)-endothelial cell interaction of arterialized syngeneic orthotopic liver transplants in Lewis rats. Livers were preserved for 24 hr in University of Wisconsin solution and flushed immediately before reperfusion with either Ringer's lactate (group A: n = 7), Ringer's lactate + 0.2 mmol/liter adenosine (group B: n = 6), or Carolina rinse (group C: n = 7). Microvascular perfusion and WBC accumulation were assessed by intravital fluorescence microscopy. In group C, acinar perfusion was significantly improved, accompanied by a lower percentage of nonperfused sinusoids 1 hr after reperfusion (mean +/- SEM: 26 +/- 2% [group A], 21 +/- 2% [B], 11 +/- 1% [C], P < 0.01 for C vs. A or B). In addition, Carolina rinse and, to a lesser extent, adenosine rinse reduced the number of WBC sticking in sinusoids and postsinusoidal venules. Better graft function in group C was indicated by increased bile flow during the observation period of 90 min after reperfusion (0.5 +/- 0.3 ml/100 g liver [group A], 1.5 +/- 0.7 [B], 3.7 +/- 0.6 [C], P < 0.01 for C vs. A or B). We conclude that Carolina rinse is able to improve early excretory hepatocellular function, microvascular perfusion, and intrahepatic WBC accumulation after prolonged cold ischemia and reperfusion, but adenosine is unlikely to be the key component of this rinse solution.     

Impact of N-acetylcysteine on the hepatic microcirculation after orthotopic liver transplantation

Recent observations showed an improvement of hepatic macro- and microhemodynamics as well as survival rates after warm ischemia of the liver following treatment with N-acetylcysteine (NAC). In this study we assessed the influence of NAC on the hepatic microcirculation after orthotopic liver transplantation (OLT) using intravital fluorescence microscopy. OLT with simultaneous arterialization was performed in 16 male Lewis rats following cold storage in University of Wisconsin solution for 24 hr. Within the experimental group (n = 8) donors received NAC (400 mg/kg) 25 min before hepatectomy. In addition, high-dose treatment of recipients with NAC (400 mg/kg) was started with reperfusion. Control animals (n = 8) received an equivalent amount of Ringer's solution. Intravital fluorescence microscopy was performed 30-90 min after reperfusion assessing acinar and sinusoidal perfusion, leukocyte-endothelium interaction, and phagocytic activity. Treatment with NAC reduced the number of nonperfused sinusoid from 52.4 +/- 0.8% to 15.7 +/- 0.5% (p = 0.0001) (mean +/- SEM). Furthermore, we achieved a significant reduction of leukocytes adhering to sinusoidal endothelium (per mm2 liver surface) from 351.9 +/- 13.0 in controls to 83.6 +/- 4.2 in the experimental group (P = 0.0001). In postsinusoidal venules, treatment with NAC decreased the number of sticking leukocytes (per mm2 endothelium) from 1098.5 +/- 59.6 to 425.9 +/- 37.7 (P = 0.0001). Moreover, bile flow was significantly increased after therapy with NAC (4.3 +/- 1.2 vs. 2.2 +/- 0.7 ml/90 min x 100g liver) (P < 0.05). Phagocytic activity was not influenced by application of NAC. We conclude that high-dose therapy with NAC in OLT attenuates manifestations of microvascular perfusion failure early after reperfusion and should be considered as a means to reduce reperfusion injury.     

A comparison of the efficacy and tolerability of dorzolamide and acetazolamide as adjunctive therapy to timolol. Oral to Topical CAI Study Group

BACKGROUND: Recent observations provide evidence that complement is involved in the pathophysiology of ischemia/reperfusion injury. In this study, we assessed the impact of complement inhibition on hepatic microcirculation and graft function using a rat model of liver transplantation. METHODS: Arterialized orthotopic liver transplantation was performed in Lewis rats after cold preservation (University of Wisconsin solution, 4 degrees C, 24 h). Eight animals received the physiological complement regulator soluble complement receptor type 1 (sCR1) intravenously 1 min before reperfusion. Controls received Ringer's solution (n=8). Microvascular perfusion, leukocyte adhesion, and Kupffer cell phagocytic activity were studied 30-100 min after reperfusion by in vivo microscopy. RESULTS: Microvascular perfusion in hepatic sinusoids was improved in the sCR1 group (87+/-0.7% vs. 50+/-1%; P < 0.001). The number of adherent leukocytes was reduced in sinusoids (68.3+/-4.7 vs. 334.1+/-15.8 [adherent leukocytes per mm < or = liver surface]; P < 0.001) and in postsinusoidal venules after sCR1 treatment (306.6+/-21.8 vs. 931.6+/-55.9 [adherent leukocytes per mm < or = endothelial surface]; P < 0.001). Kupffer cell phagocytic activity was decreased in the sCR1 group compared to controls. Postischemic bile production reflecting hepatocellular function was increased by almost 200% (P = 0.004) after complement inhibition. Plasmatic liver enzyme activity was decreased significantly upon sCR1 treatment, indicating reduced parenchymal cell injury. CONCLUSIONS: Our results provide further evidence that the complement system plays a decisive role in hepatic ischemia/reperfusion injury. We conclude that complement inhibition by sCR1 represents an effective treatment to prevent reperfusion injury in liver transplantation.     

E-selectin appears in nonischemic tissue during experimental focal cerebral ischemia

BACKGROUND AND PURPOSE: E-selectin participates in leukocyte-endothelial adhesion and the inflammatory processes that follow focal cerebral ischemia and reperfusion. The temporal and topographical patterns of microvascular E-selectin presentation after experimental focal cerebral ischemia are relevant to microvascular reactivity to ischemia. METHODS: The upregulation and fate of E-selectin antigen during 2 hours of middle cerebral artery occlusion (n = 4) and 3 hours of occlusion with reperfusion (1 hour, n = 4; 4 hours, n = 6; 24 hours, n = 6) were evaluated in the nonhuman primate. E-selectin and E:P-selectin immunoreactivities were semiquantitated with the use of computerized light microscopy video imaging and laser confocal microscopy. RESULTS: Three patterns of microvascular E-selectin expression, defined by the antibody E-1E4, were confirmed by complete elimination of E-1E4 binding after incubation with soluble recombinant human E-selectin: (1) Low immunoperoxidase intensity was observed in ischemic microvessels at 2 hours of occlusion extending to 4 hours of reperfusion (E-selectin/laminin = 0.32 +/- 0.10). (2) A significant fraction of ischemic microvessels displayed high-intensity E-selectin signal by 24 hours of reperfusion (0.61 +/- 0.17) compared with control and nonischemic tissues (2P < .003). (3) In the contralateral nonischemic basal ganglia and other nonischemic tissues, low but significant E-selectin levels appeared by 24 hours of reperfusion (2P = .0005). The latter were further confirmed by an E:P-selectin immunoprobe. CONCLUSIONS: E-selectin antigen is distinctively and significantly upregulated in nonhuman primate brain after focal ischemia and reperfusion. The late appearance of E-selectin in nonischemic cerebral tissues suggests stimulation by transferable factors generated during brain injury.     

Changes in vessel ultrastructure during ischemia and reperfusion of rabbit hindlimb: implications for therapeutic intervention

Peripheral ischemia was induced in the rabbit by occlusion of the left iliac artery for 6 hr, followed by 24 hr of reperfusion. Biochemical and morphological investigations were performed to evaluate the extent of vascular and tissue injury. Blood samples for plasma enzyme determinations (creatine kinase (CK) and lactate dehydrogenase (LDH) activities) were obtained at times t = 0, t = 6, t = 30 hr. Plasma CK and LDH activities in ischemic animals were approximately twice as high as those in sham-operated animals at the end of reperfusion, although no difference was observed at the end of the period of ischemia. Morphological and morphometric analysis of extensor digitorum longus muscle from ischemic animals showed a reduction in the number of patent capillary vessels per muscle fiber (1.54 +/- 0.1 and 1.04 +/- 0.09, P < 0.05, in sham and ischemic groups, respectively; mean +/- SEM). In addition, the number of microvilli on endothelial surfaces were considerably increased in the ischemic group (0.14 +/- 0.02 and 0.41 +/- 0.01 microns -2, P < 0.001, in sham and ischemic groups, respectively). A great number of adhered leucocytes were found on the vessel surface with some leucocytes having migrated through the vessel wall. Microcirculatory damage was accompanied by the formation of microthrombi which sometimes occluded the entire vessel lumen. The infusion of 1 mg/kg/hr of cloricromene for 6 hr prevented ischemic injury in microvessels and also prevented swelling of muscle mitochondria. In the treated group the number of patent capillaries per muscle fiber was very similar to that found in sham-operated animals (1.49 +/- 0.08; P < 0.01 vs. ischemic control). In conclusion, several different cell types are involved in the pathophysiological changes which occur in microvessels during ischemia/reperfusion injury. Pharmacological interventions, which inhibit the interactions of blood cells with endothelium, may be of value in the treatment of peripheral ischemia/reperfusion injury.     

Hemodynamics in nailfold capillaries of patients with systemic scleroderma: synchronous measurements of capillary blood pressure and red blood cell velocity

There is increasing evidence that endothelial damage occurs at a very early stage during the course of systemic scleroderma. Endothelial damage is accompanied by impaired microvascular function, which has clearly failed in patients with systemic scleroderma, as evidenced by necrosis of the fingertips in severe cases. We investigated two important determinants of microvascular function, namely capillary blood pressure and capillary red blood cell velocity, simultaneously in the same capillary. In patients with systemic scleroderma and in healthy volunteers matched for age and sex, capillary blood pressure was measured by direct cannulation and capillary red blood cell velocity by video microscopy. Capillary blood pressure and capillary red blood cell velocity were significantly lower in patients (14.27 +/- 4.34 mmHg, 230 +/- 310 microm per s) than in healthy controls (19.06 +/- 3.69 mmHg, p < 0.008, and 910 +/- 240 microm per s, p < 0.003) at an ambient temperature of 22 degrees C, whereas no significant difference in skin temperature was observed (23.7 +/- 0.9 degrees C vs 24.7 +/- 1.9 degrees C) and no occlusion of finger arteries was detected. Capillary blood pressure in enlarged capillaries did not differ from that in normal-shaped capillaries in the patients (correlation of diameter and capillary blood pressure, R2 = 0.04), which was also the case with capillary red blood cell velocity (R2 = 0.13). Capillary pulse pressure amplitude and capillary red blood cell velocity showed a strong correlation (R2 = 0.81), suggesting that the pressure gradient across the capillary loop, which is the driving force for capillary red blood cell velocity, was mainly dependent on precapillary resistance. These observations reflect the inadequate microvascular function in systemic scleroderma, which may be due mainly to a pathophysiologic functional increase in precapillary resistance, even at comfortable ambient temperatures.     

Disturbance of peripheral microvascular function in congestive heart failure secondary to idiopathic dilated cardiomyopathy

OBJECTIVES: Previous studies of peripheral microvascular function in human heart failure have concentrated on changes in flow, and there is little information concerning the impact of heart failure on the principal determinants of transcapillary fluid exchange. This study investigated whether alterations in capillary pressure and microvascular fluid permeability can be detected in subjects with idiopathic dilated cardiomyopathy. METHODS: Finger nailfold capillary pressure and calf capillary filtration coefficient (CFC) were measured in parallel studies of two overlapping groups of 12 non-oedematous subjects with idiopathic dilated cardiomyopathy and mild to moderate heart failure and in age- and sex-matched healthy controls. Capillary pressure was measured by direct cannulation using an electronic resistance feedback servonulling technique, and CFC by mercury-in-silastic strain gauge plethysmography using a modification of the technique which avoids assumptions concerning isovolumetric venous pressure. RESULTS: Following correction for differences in skin temperature, capillary pressure was lower in the subjects with heart failure (P = 0.02). Both CFC and isovolumetric venous pressure were greater in the subjects with heart failure than in controls (3.4 +/- 0.9 vs. 2.6 +/- 0.7 ml.min-1.mmHg-1.100 ml-1, P = 0.03; 27.1 +/- 8.4 vs. 17.2 +/- 7.2 mmHg, P = 0.01). CONCLUSIONS: These data suggest that factors other than changes in arterial inflow and venous outflow pressures are likely to play an important role in the disruption of microvascular homeostasis which occurs in heart failure. Changes in capillary hydraulic conductance may contribute to the pathogenesis of oedema.     

Effects of mixed ETA and ETB-receptor antagonist (Ro-47-0203) on hepatic microcirculation after warm ischemia

There is evidence that endothelin (ET) is involved in disturbances of the hepatic microcirculation after warm ischemia. In this study we investigated the influence of a mixed ETA-, ETB-receptor antagonist (Bosentan) on ischemia-reperfusion damage of the liver by means of intravital fluorescence microscopy (IVM). Clamping of the left liver lobe (= warm ischemia) was performed in 16 male Wistar rats for 70 min. The treatment group (N = 8) received 15 mg/kg Bosentan (Ro-47-0203) 1 min prior to reperfusion. Controls (N = 8) received an equivalent amount of Ringer's solution. Between 20 and 90 min after reperfusion, leukocyte-endothelial cell interactions in sinusoids and postsinusoidal venules as well as perfusion of hepatic acini were studied. Application of Bosentan improved sinusoidal blood flow, attenuated manifestations of microvascular perfusion failure, and decreased the number of rolling leukocytes in postsinusoidal venules. Our results provide further evidence that ET is involved in postischemic impairment of hepatic microhemodynamics during reperfusion.     

Myoelectric activity during small bowel allograft rejection

The effect of rejection on myoelectric activity of an orthotopically transplanted small intestinal segment (group I, N = 14) was studied. Electrodes were placed on grafts and recipient small bowel. Isografts (group II, N = 5) and native bowel (group III, N = 5) served as controls. The first morphological signs of rejection were seen on day 6 and steadily progressed until day 11, when the cellular infiltrate involved all layers of the bowel wall. Slow-wave frequencies remained unchanged throughout the observation period. No difference was detectable between grafts (group I: 31.9 +/- 1.65; group II: 31.36 +/- 0.7) and native bowel after transection (group I: 32.16 +/- 1.78; group II: 31.50 +/- 1.01), which was different (P = 0.0001) from intact bowel of group III animals (38.4 +/- 0.81). Irregular MMCs were detectable in grafts from day 5 on and replaced after food intake by random spiking activities. At day 8, spiking activities disappeared in allografts, which showed a still preserved mucosal architecture, while slow-wave activities continued. These findings demonstrate that intestinal allografts during rejection develop paralysis before mucosal destruction is established, which might be of clinical relevance.     

Eosinophil surface antigen expression and cytokine production vary in different ocular allergic diseases

OBJECTIVES: Improving methods of donor heart preservation may permit prolonged storage and remote procurement of cardiac allografts. We hypothesized that continuous, sanguineous perfusion of the donor heart in the beating, working state may prolong myocardial preservation. METHODS: We developed a portable perfusion apparatus for use in donor heart preservation. Contractile, metabolic, and vasomotor functions were monitored simultaneously in an isolated swine heart. The metabolic state was monitored by myocardial tissue pH. Vasomotor function was assessed in isolated coronary ring chambers. Hearts were randomized into 3 groups: group I (n = 5), cardioplegic arrest, 12-hour storage at 4 degrees C with modified Belzer solution, and 2-hour sanguineous reperfusion in the working state; group II (n = 6), 12-hour continuous perfusion in the beating working state, 30 minutes of arrest (to simulate re-implantation time), and 2 hours of reperfusion, as above; group III (n = 7), coronary ring control hearts. RESULTS: At 2 hours of reperfusion, left ventricular developed pressure in group II was higher than in group I (mean +/- standard deviation: 90 +/- 6 mm Hg, 53 +/- 15 mm Hg, P = .005). Significantly less myocardial edema was observed in group II than in group I (73% +/- 4%, 80% +/- 1% water content, P = .01). Significantly less myocardial acidosis was noted in group II than in group I during preservation (pH 7.3 +/- 0.01, 6.1 +/- 0.03, P < .001) and reperfusion (pH 7.3 +/- 0.008, 6.8 +/- 0.05, P < .001). Coronary endothelial vasomotor function was better preserved in group II than in group I as evidenced by dose-response relaxation of coronary rings to 10(-8) mol/L bradykinin (37%, 55% delta baseline, P = .01). CONCLUSION: This new method extends the current preservation limit and avoids time-dependent ischemic injury, thereby allowing for distant procurement of donor organs.     

A CD18 antibody prevents lung injury but not hypotension after intestinal ischemia-reperfusion

Antibodies to the neutrophil CD18 integrin have been shown to ameliorate the local effects of intestinal ischemia and reperfusion (I/R). In addition to local mucosal injury, intestinal I/R results in systemic hypotension and injury to the lungs with lung leukosequestration. This study tests the effect of a CD18 monoclonal antibody on the hypotension and lung injury after intestinal I/R. In anesthetized rabbits, the superior mesenteric artery was clamped for 60 min followed by 3 h of reperfusion. Animals were treated with saline, an anti-CD18 monoclonal antibody (R15.7 MAb), or nonspecific immunoglobulin G. Another non-ischemic group were sham controls. Neutrophil sequestration was assessed by measure of lung myeloperoxidase (MPO) and permeability by lung-to-blood concentration ratio of 125I-labeled bovine serum albumin and wet-to-dry weight ratio. Immediately after reperfusion, mean arterial pressure fell to 49 +/- 2.1 mmHg and remained at this level. The hypotension was unaffected by treatment with R15.7 MAb. Thirty minutes after reperfusion, the circulating white blood cell count fell to 2.91 +/- 0.53 x 10(3)/mm3 vs. sham 6.40 +/- 0.66 x 10(3)/mm3 (P < 0.05). Treatment with R15.7 MAb prevented this fall in white blood cell count (5.75 +/- 1.59 x 10(3)/mm3). At 3 h of reperfusion in saline-treated animals there was increased MPO, 74.8 +/- 4.9 U/g vs. 42.0 +/- 4.8 U/g in sham animals (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ischemic preconditioning on adenine nucleotide levels of graft lung from canine donor

Twelve canine left lung allotransplantation were performed. In the ischemic preconditioning group (Group IP, n = 6), left donor lung was preconditioned with 10 min ischemia followed by 15 min reperfusion using the occuluding left hilum before resection and cold perfusion. The control group (Group C, n = 6) underwent the same treatment but without ischemic preconditioning. Adenine nucleotides (ATP, ADP, AMP) of the donor lung tissue were measured using rHPLC after 2 hr of resection and cold perfusion with Euro-Collins solution (ECS). The results showed that contents of ATP and total adenine nucleotides (TAN) were much higher in Group IP than in Group C (322.9 +/- 61.2, 942.9 +/- 134.5 and 200.0 +/- 50.0, 668.4 +/- 59.6 mumol.g-1 respectively, P < 0.05). Histologic examination of the donor lung in Group IP showed less damage than in Group C after 2 hr of transplantation. The results suggest that IP combined with cold ECS perfusion can reduce the energy metabolism in canine donor lung.     

Modulation of retinal endothelial barrier in an in vitro model of the retinal microvasculature

The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic 'cell-columns'. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3 x 10(4) cells cm-2 of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66 cm in diameter and 1.6 cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2 x 10(6) Da; cyanocobalamin 1355 Da; sodium fluorescein 376 Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57 +/- 0.57 x 10(-5) cm sec-1 for cyanocobalamin and 9.29 +/- 0.78 x 10(-5) cm sec-1 for sodium fluorescein (mean +/- S.E.M., n = 39). Permeability did not increase over 2 hr of cell-column perfusion. Permeability was decreased by 1 micron isoproterenol (n = 3) and increased by 1 microgram ml-1 cytochalasin D (n = 5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.     

Metalloregulated expression of the ars operon

Myocardial dysfunction after cardiac operations might be influenced by altered myocardial perfusion in the postoperative period. To investigate possible alterations in vascular reactivity, in vitro coronary microvascular responses were examined after ischemic cardioplegia with use of a porcine model of cardiopulmonary bypass. Since myocardial perfusion is primarily regulated by arteries less than 200 microns in diameter, these vascular segments were examined. After 1 hour of ischemic arrest with cold crystalloid cardioplegia and 1 hour of reperfusion, microvessels (100 to 190 microns in diameter) were pressurized in a no-flow state, preconstricted by 30% to 60% of the baseline diameter with acetylcholine, and examined with video microscopic imaging and electronic dimension analysis. Endothelium-dependent relaxations to bradykinin (55% +/- 13% versus 99% +/- 1% = maximum relaxation of the preconstricted diameter in cardioplegia-reperfusion vessels versus control vessels, respectively; p < 0.05) and the calcium ionophore A 23187 (33% +/- 6% versus 90% +/- 4%; p < 0.05) were markedly impaired while endothelium-independent relaxation to sodium nitroprusside was similar to control value. After 1 hour of ischemic cardioplegia without reperfusion, endothelium-dependent relaxation was only slightly affected. Transmission electron microscopy showed minimal endothelial damage after ischemic cardioplegia and reperfusion. These findings have important implications regarding coronary spasm and cardiac dysfunction after cardiac operations.     

Suppressed anti-aggregating and cGMP-elevating effects of sodium nitroprusside in platelets from patients with stable angina pectoris

Short bowel syndrome (SBS) in the newborn results in limited intestinal absorptive capacity, leading especially to fatty acid (FA) malabsorption. It is unknown whether adaptation occurs in time in FA absorption, and whether this adaptation is chain-length dependent. The aid of the present study was to prospectively evaluate FA absorption and excretion during SBS in the newborn. Twenty-one neonates who underwent small bowel resection (of variable length) for various reasons (necrotizing enterocolitis, intestinal atresia, meconium peritonitis, cloacal extrophy, etc) were studied. Eight neonates had SBS, defined as a small bowel remnant of less than 50% of the original small bowel length related to gestational age. The mean remaining small bowel length in the SBS group was 34% (24% to 42%). The non-SBS control group consisted of 13 neonates who had only minor small bowel resections. The mean remaining bowel length for the non-SBS group was 95% (70% to 100%). The results show that the total fractional excretion of FA (FE-FA) at 2 weeks and 1, 2, 3, and 4 months postsurgery was 51% +/- 37%, 33% +/- 24%, 51% +/- 65%, 53% +/- 27%, and 7% +/- 2% in patients with SBS, versus 12% +/- 8%, 24% +/- 10%, 9% +/- 3%, 8% +/- 3% and 17% +/- 14% in the non-SBS controls, respectively (P < .05 by ANOVA). There appeared to be an amelioration in time in FA absorption, especially in the SBS group, after 3 months. FE-FA was chain-length related, being considerably less for C10 and C12 than for C14 and longer amounts. An amelioration of absorption occurred in the SBS patients, especially with the longer-chain FA. On the basis of the study data, the authors conclude that in the initial adaptation phase shorter chain lengths are better absorbed than longer chain lengths; however, in the latter FA group, substantial adaptation occurs with time.     

Preservation of pig liver allografts after warm ischemia: normothermic perfusion versus cold storage

Warm ischemia is known to induce substantial damage to the liver parenchyma. With respect to clinical liver transplantation, the tolerance of the liver to warm ischemia and the preservation of these organs have not been studied in detail. In isolated reperfused pig livers we proceeded according to the following concept: Livers were subjected to 1 or 3 h of warm ischemia. Subsequently, these organs were preserved by either normothermic perfusion or cold storage (histidine-tryptophan-alpha-ketoglutarate, HTK) for 3 h each. After storage, liver function was assessed in a reperfusion circuit for another 3 h. Parameters under evaluation were bile flow, perfusion flow, oxygen consumption, enzyme release into the perfusate (creatine kinase, glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase, and glutamic pyruvic transaminase), and histomorphology. Damage to the liver was lowest after warm ischemia of 1 h. The results after cold storage were superior to those after normothermic perfusion (GOT: 3.2 +/- 0.3 and 2.6 +/- 0.2 U/g liver; cumulative bile production: 14.7 +/- 2.1 and 9.4 +/- 1 ml, respectively; P < 0.05). In contrast, we found substantial damage at the end of reperfusion in livers undergoing 3 h of warm ischemia under both preservation techniques with severe hepatocellular pyknoses and essentially altered nonparenchymal cells. The results suggest that pig livers undergoing 1 h of warm ischemia and cold storage for 3 h with HTK solution may lead to functioning after transplantation.     

Energy metabolism and tissue blood flow as parameters for the assessment of graft viability in rat small bowel transplantation

The assessment of small bowel graft viability by means of energy metabolism and tissue blood flow was investigated and compared with pathological findings. Syngeneic heterotopic small bowel transplantations were performed using male Lewis rats, which were divided into four groups according to the duration of cold preservation in University of Wisconsin (UW) solution; 6-, 12-, 24-, and 48-hour groups. The adenine nucleotide metabolism, the tissue blood flow, and the pathological profiles of the grafts were all compared among the groups. The adenosine triphosphate (ATP) levels at the end of cold storage and at 30 minutes after reperfusion, as well as the total adenine nucleotide (TAN) levels at the end of cold storage, before reperfusion, and at 30 minutes after reperfusion were significantly lower in the 48-hour group than those in the other groups, and the blood flow level at reperfusion was significantly lower in the 48-hour group than that in the others. Histological damage after reperfusion extended deep into the crypt layer in the 48-hour group but was confined to within villi in the other groups. These results suggest that the tissue ATP, TAN, and the blood flow levels are considered useful parameters for the assessment of small bowel graft viability.     

Ischemia-reperfusion induced microvascular dysfunction in skeletal muscle: application of intravital video microscopy

Video microscopy of red cell flow in capillaries at the surface of skeletal muscle provided the opportunity to quantitate ischemia-reperfusion (I-R) induced microcirculatory changes, in vivo. Extensor Digitorum Longus (EDL) muscles of 22 male Wistar rats (300-400 g), anesthetized with sodium pentobarbital (Somnotol, 65 mg kg,-1 IP), were used to measure the number of perfused capillaries (CDper: mm-1) crossing lines drawn perpendicular to the muscle axis, and red blood cell velocity (VRBC: mm/s) within individual capillaries from controls (n = 6), and after 2 hr (n = 4), 3 hr (n = 4), and 4 hr (n = 5) of no-flow ischemia with the muscle temperature maintained at its normal value of 32 degrees C. Ischemia was induced by tightening a tourniquet placed around the limb above the EDL muscle. Measurements were made after 30, 60, and 90 min of reperfusion. To test the usefulness of this skeletal muscle model for evaluating proposed interventions in I-R, the effect of hypothermia (24 degrees C) on the microcirculation following 4 hr ischemia (n = 3) was measured. Edema formation was estimated from the wet/dry weight ratio of the ischemic and contralateral control EDL muscles. Capillary perfusion at the surface of the control muscles was remarkably stable over the 5 hr period studied, while significant changes occurred following the ischemic periods. Significantly lower CDper was measured 30 min following all periods of normothermic ischemia. However, unlike the 2 and 4 hr ischemic periods 3 hr normothermic ischemia resulted in a progressive decline in CDper throughout the reperfusion period. VRBC showed evidence of a hyperemic response following 2 hr normothermic ischemia (control: 0.12 mm/s +/- 0.19 compared to 0.26 mm/s +/- 0.03 following 90 min reperfusion; mean +/- sem). However, no such hyperemia was measured following either 3 or 4 hr normothermic ischemia (i.e., 3 hr control: 0.24 mm/s +/- 0.01 compared to 0.07 mm s +/- 0.003 following 90 min reperfusion). In fact, VRBC was essentially zero 90 min following 4 hr normothermic ischemia (0.01 mm/s +/- 0.01). However, when the muscle was allowed to cool to 24 degrees C during 4 hr ischemia no significant change in either VRBC or CDper was measured compared to pre-ischemic controls. Evidence of edema was found after 3 and 4 hr normothermic ischemia. This study establishes a skeletal muscle model of I-R, which may be useful in testing hypotheses regarding mechanisms of I-R injury, and effectiveness of proposed treatments of I-R.