Glucagon-like peptide-1 (GLP-1), immediately prior to reperfusion, decreases neutrophil activation and reduces myocardial infarct size in rodents.

Glucagon-like peptide-1 (GLP-1) is an incretin that has glucoregulatory effects as well as protective effects in a variety of tissues, including the heart. We hypothesized that GLP-1 may have a direct effect on neutrophils (PMNs) after myocardial ischemia, to ameliorate reperfusion injury. Deeply anesthetized Sprague-Dawley rats underwent 30 min of left coronary artery occlusion followed by 120 min of reperfusion. Immediately prior to reperfusion, rats were treated with either GLP-1 (human rGLP-1, 30 pM/kg/min) or PBS as placebo. GLP-1 significantly decreased myocardial infarct size [73.2±11.7% INF/AAR in PBS (n=4) vs. 15.7 ±5.52% INF/AAR in GLP-1-treated animals (n=5), p<0.05], PMN activation in blood in vivo (fMLP-stimulated CD11b surface expression: PBS 2.78±1.14 vs. GLP-1 1.7±0.21, TFI, p<0.05), and accumulation in myocardium (PBS: 6.52±0.31 vs. GLP-1: 4.78±0.90, n=4-6 animals/group, p<0.05). In addition, we found that GLP-1 mitigated PMN CD11b surface expression in whole rat blood in vitro, an effect that was abolished by GLP-1 receptor blockade (PBS 6.52±0.31 vs. GLP-1 4.78±0.90, TFI, p<0.05). These findings suggest that one mechanism by which GLP-1 decreases reperfusion injury may be the attenuation of PMN-mediated reperfusion injury.

Pentoxifylline reduces coronary leukocyte accumulation early in reperfusion after cold ischemia.

BACKGROUND: Ischemia/reperfusion injury can complicate recovery in cardiac operations. Ischemia induces endothelial dysfunction, which may contribute to leukocyte accumulation during reperfusion. Leukocyte-mediated injury may then occur. Using intravital microscopy we previously reported increased leukocyte retention in coronary capillaries and venules during early reperfusion during warm ischemia/reperfusion. In this study we investigated whether cold cardioplegic protection would limit leukocyte sequestration in coronary microvessels early in reperfusion. Pentoxifylline (PTX) has antiinflammatory effects and may limit endothelial dysfunction during ischemia/reperfusion. The effect of cardioplegia modification with PTX was also examined. METHODS: Isolated rat hearts were subjected to 90 minutes of 4 degrees C ischemia after arrest with cardioplegia. Hearts were reperfused with diluted whole blood containing fluorescent-labeled leukocytes. Leukocyte retention in coronary microvessels was observed with intravital microscopy. Three groups were studied, nonischemic control, cold ischemia, and PTX-modified cold ischemia. RESULTS: In coronary capillaries, leukocyte trapping was nearly doubled in unmodified cold ischemia versus control. PTX modification significantly reduced leukocyte accumulation. In coronary venules, greater leukocyte adhesion was observed in unmodified cold ischemia compared to nonischemic controls. PTX modification significantly reduced leukocyte adhesion. CONCLUSIONS: Cold cardioplegia did not prevent leukocyte retention in the coronary microcirculation early in reperfusion. PTX modification of cardioplegia significantly reduced leukocyte sequestration in coronary capillaries and venules. Preserving endothelial function during ischemia may limit leukocyte accumulation and ischemia/reperfusion injury after cardiac operation.

Diabetes enhances leukocyte accumulation in the coronary microcirculation early in reperfusion following ischemia.

BACKGROUND: Diabetic hearts are particularly vulnerable to ischemia-reperfusion injury. For leukocytes to participate in ischemia-reperfusion injury, they must first sequester in the microcirculation. The aim of this study was to determine, by direct observation, if early leukocyte deposition was increased in the diabetic coronary microcirculation early in reperfusion following myocardial ischemia. METHODS: Non-diabetic and streptozotocin (STZ)-induced diabetic rat hearts, subjected to 30 min of 37 degrees C, no-flow ischemia, were initially reperfused with blood containing labeled leukocytes. The deposition of fluorescent leukocytes in coronary capillaries and venules was directly visualized and recorded using intravital fluorescence microscopy. In addition, flow cytometry was used to measure CD11b adhesion molecule expression on polymorphonuclear (PMN) leukocytes from non-diabetic and STZ-diabetic rats. RESULTS: In the non-diabetic, control hearts, early in reperfusion, leukocytes trapped in coronary capillaries and adhered to the walls of post-capillary venules. In the diabetic hearts, leukocyte trapping in capillaries and adhesion to venules were both significantly increased (P<0.05). PMN CD11b expression was also significantly increased in the diabetic blood compared to the non-diabetic blood (P<0.05). CONCLUSIONS: Early in reperfusion following myocardial ischemia, leukocytes rapidly accumulate in greater numbers in the coronary microcirculation of the diabetic heart by both trapping in coronary capillaries and by adhering to venules. The enhanced retention of leukocytes in the diabetic coronary microcirculation increases the likelihood of inflammation-mediated reperfusion injury and may explain, in part, the poor recovery of diabetic hearts from an ischemic event.

Microvascular perfusion and transport in the diabetic heart.

Diabetes is a chronic disease of metabolic dysfunction that is increasing world-wide. The hyperglycemia associated with diabetes causes significant protein alterations and an oxidative stress. In the heart, all cell types are affected by diabetes: the myocyte, the vasculature and the blood cells. Four out of five diabetics die from ischemic heart disease and stroke, suggesting that the diabetic is quite vulnerable to ischemic injury. It is important to understand the pathophysiologic challenges that occur in the diabetic heart in order to develop thoughtful treatments to limit this serious complication. This review focuses on the anatomical and functional alterations that occur in the diabetic circulation of the heart, with emphasis on the coronary microcirculation. Coronary microvascular dysfunction combined with blood cellular alterations are presented to explain the amplified oxidative stress that occurs in the diabetic heart under ischemic conditions.

Leukocyte accumulation and hemodynamic changes in the cerebral microcirculation during early reperfusion after stroke.

BACKGROUND AND PURPOSE: Leukocytes contribute to cerebral ischemia-reperfusion injury. However, few experimental models examine both in vivo behavior of leukocytes and microvascular rheology after stroke. The purpose of the present study was to characterize patterns of leukocyte accumulation in the cerebral microcirculation and to examine the relationship between leukocyte accumulation and microcirculatory hemodynamics after middle cerebral artery occlusion and reperfusion (MCAO-R). METHODS: Male rats (250 to 350 g) were anesthetized and ventilated. Tail catheters were inserted for measurement of arterial blood gases and administration of drugs. Body temperature was maintained at 37 degrees C. Animals were subjected to 2 hours of MCAO by the filament method. A cranial-window preparation was performed, and the brain was superfused with warm, aerated artificial cerebrospinal fluid. Reperfusion was initiated by withdrawing the filament, and the pial microcirculation was observed by use of intravital fluorescence microscopy. Leukocyte accumulation in venules, arterioles, and capillaries; leukocyte rolling in venules; and leukocyte venular shear rate were assessed during 1 hour of reperfusion. RESULTS: We found significant leukocyte adhesion in cerebral venules during 1 hour of reperfusion after 2 hours of MCAO. Leukocyte trapping in capillaries and adhesion to arterioles after MCAO-R tended to increase compared with controls, but the increase was not significant. We also found that shear rate was significantly reduced in venules during early reperfusion after MCAO. CONCLUSIONS: A model using the filament method of stroke and fluorescence microscopy was used to examine white-cell behavior and hemodynamics in the cerebral microcirculation after MCAO-R. We observed a significant increase in leukocyte rolling and adhesion in venules and a significant decrease in blood shear rate in the microcirculation of the brain during early reperfusion. Leukocytes may activate and damage the blood vessels and surrounding brain cells, which contributes to an exaggerated inflammatory component to reperfusion. The model described can be used to examine precisely blood cell-endothelium interactions and hemodynamic changes in the microcirculation during postischemic reperfusion. Information from these and similar experiments may contribute to our understanding of the early inflammatory response in the brain during reperfusion after stroke.

Fucoidin reduces coronary microvascular leukocyte accumulation early in reperfusion.

BACKGROUND: Leukocytes rapidly accumulate in the heart early in reperfusion after ischemia, contributing to reperfusion injury. The purpose of this study was to determine whether treatment with the selectin blocker fucoidin (FCN) would attenuate early leukocyte retention in coronary venules and capillaries during low flow reperfusion. METHODS: Isolated rat hearts subjected to 30 minutes of 37 degrees C, no-flow ischemia were initially reperfused with blood containing labeled leukocytes, followed by reperfusion with a Krebs red cell solution. The deposition of leukocytes in coronary capillaries and venules was observed using intravital microscopy. Three groups were studied: nonischemic control hearts, untreated postischemic hearts reperfused at low flow, and postischemic hearts reperfused at low flow, where both the hearts and the blood reperfusate were pretreated with FCN (0.36 mg/mL blood). RESULTS: In the ischemia-reperfusion group, we observed a rapid and significant increase in leukocyte accumulation in both capillaries and venules. Treatment with FCN significantly reduced the leukocyte accumulation in both capillaries and venules (p<0.05). In addition, FCN significantly reduced the persistence of leukostasis in both capillaries and venules, indicating that FCN affected a transient adhesion process. CONCLUSIONS: These results suggest that the selectin family of leukocyte-endothelial cell adhesion proteins mediates the initial retention of leukocytes in both coronary capillaries and venules during reperfusion. Selectin blockade may be effective in reducing the contribution of leukocytes to early reperfusion injury.

The blood contribution to early myocardial reperfusion injury is amplified in diabetes.

Cardiovascular disease is excessive in diabetes, and blood cell function is altered. It is not clear, however, if alterations in the blood contribute to the excessive cardiovascular complications of this disease. In this study, we compared the contribution of nondiabetic and diabetic blood to myocardial reperfusion injury. The recovery of cardiac contractile function following no-flow ischemia was studied in isolated diabetic and nondiabetic rat hearts perfused with diabetic or nondiabetic diluted whole blood. Hearts were isolated from 10- to 12-week-old diabetic (streptozotocin, 65 mg/kg, i.v.) and nondiabetic rats and perfused with a Krebs-albumin-red cell solution (K2RBC, Hct 20%). After a 30-min pre-ischemic control period, during which cardiac pump function was evaluated, diabetic and nondiabetic hearts were perfused for 5 min with diluted whole blood (DWB; Hct 20%) collected from either diabetic or nondiabetic donor animals. Coronary flow was then stopped and the hearts subjected to 30 min of no-flow ischemia. Following ischemia, the hearts were reperfused with the K2RBC perfusate. Cardiac contractile function was evaluated throughout the 60-min reperfusion period. Six groups were studied: diabetic and nondiabetic hearts perfused before ischemia with either K2RBC, nondiabetic DWB (NDDWB), or diabetic DWB (DDWB). Perfusion with DWB prior to ischemia impaired the recovery of contractile function in all cases. The impairment to recovery was greater with DDWB than with NDDWB. Although diabetic hearts perfused with K2RBC throughout recovered quite well, the effect of DDWB perfusion in the diabetic hearts was dramatic. In an effort to determine why diabetic blood impaired functional recovery, measures of blood filterability and the generation of reactive oxygen species (ROS) were made. We found that diabetic blood was less filterable than nondiabetic blood; that is, the diabetic blood cells tended to plug the 5-microm filter pores more readily than the nondiabetic blood cells. Also, we found that the diabetic blood was capable of generating significantly greater ROS (oxygen free radicals) than nondiabetic blood (P < 0.05). These findings suggest that the blood contribution to myocardial reperfusion injury is amplified in diabetes. A tendency for diabetic blood cells to plug capillary-sized pores and show enhanced oxygen free radical production may account for the excessive contribution of diabetic blood to reperfusion injury in the heart.

Low-flow reperfusion after myocardial ischemia enhances leukocyte accumulation in coronary microcirculation.

During early reperfusion after myocardial ischemia, the mechanisms responsible for leukocyte accumulation in the heart are unclear. We examined the effects of reducing coronary blood flow during reperfusion on leukocyte accumulation in coronary capillaries and postcapillary venules. Isolated rat hearts were perfused for 30 min and then subjected to 30 min of 37 degrees C, no-flow ischemia. The deposition of fluorescently labeled leukocytes was observed directly in coronary capillaries and venules using intravital microscopy after 5, 20, and 35 min of reperfusion. Blood cell velocity was measured in venules after 5 min of reperfusion (R5), and shear rate (s-1) was calculated. Four groups were studied: nonischemic control (NIC) hearts and postischemic hearts reperfused at full flow (I/R100) and at 50 and 10% of full flow (I/R50 and I/R10, respectively). In I/R100 hearts, there was a significant increase in leukocyte trapping in capillaries compared with the NIC group (R5: 5.7 +/- 0.6 vs. 2.0 +/- 0.4 leukocytes/capillary field, respectively; P < 0.05). However, the increase in leukocyte adhesion to venules was not statistically significant compared with NIC (R5: 3.2 +/- 0.4 vs. 1.5 +/- 0.6 leukocytes/100-micron venule, respectively; P < 0.2). In I/R50 hearts, a further increase in leukocyte accumulation occurred in the capillaries but not in the venules. However, in I/R10 hearts, there was a statistically significant increase in both capillaries (R5: 9.2 +/- 0.8; P < 0.05) and venules (R5: 4.4 +/- 0.5; P < 0.05). When leukocyte margination in coronary venules was examined as a function of venular shear rate, a significant correlation (r = 0.99, P < 0.05) was found. These results suggest that, after ischemia, a reduction in reflow enhances leukocyte trapping in capillaries and that leukocyte adhesion in venules is inversely related to shear rate. Enhanced leukocyte accumulation may in turn increase the leukocyte contribution to early reperfusion injury in the heart.

CD18 antibody treatment limits early myocardial reperfusion injury after initial leukocyte deposition.

Following myocardial ischemia, initial reperfusion with whole blood impairs the recovery of ventricular function. The exact mechanisms underlying early myocardial reperfusion injury are not clear, but leukocytes play an important role. In this study, we tested if treating the initial blood reperfusate with a monoclonal antibody (CL26) against the leukocyte adhesion protein (CD18 would reduce the leukocyte contribution to early reperfusion injury. We reasoned that blocking CD18 would reduce the initial retention of leukocytes in the heart and thereby limit the inflammatory response. Rat hearts were isolated and perfused at constant flow with a red cell-rich solution (K2RBC). The perfusate was not recirculated. Baseline measures were made of coronary flow, perfusion pressure, and ventricular pump function. No-flow, normothermic ischemia was induced for 30 min, followed immediately by reperfusion, at the preischemic flowrate, with diluted whole blood (DWB, treated with either vehicle or CL26). Reperfusion was continued with K2RBC for 40 min more, during which postischemic measures were made. We found that the cardiac retention of leukocytes was not significantly different for the two groups, nor were the recoveries of ventricular function. Later in reperfusion (R35), the coronary blood flowrate and the coronary vascular resistances were not different; however, the recoveries of ventricular pump function were significantly different (+dP/dt @ R35 (%Pre-I): Vehicle: 27 +/- 9% (n = 8); CL26: 51 +/- 6% (n = 7); P < 0.05). Also, at R35, the voltage required to capture and pace the vehicle-treated hearts was significantly greater than the voltage required to pace the CL26 hearts (P < 0.05). Because the coronary flowrate and leukocyte retention were similar for both groups, the improved recovery observed in the CL26-treated group was not due to either improved flow or to reduced leukocyte retention. Rather, the findings suggest that the beneficial effect of antibody treatment was to attenuate step(s) in the acute inflammatory response that occurred after the initial deposition of leukocytes in the heart.

Pentoxifylline reduces leukocyte retention in the coronary microcirculation early in reperfusion following ischemia.

Using direct visualization techniques, we recently confirmed earlier histologic studies that leukocytes accumulate primarily in the coronary capillaries of ischemic hearts during early reperfusion. The purpose of this study was to determine if pentoxifylline (PTX) would reduce leukocyte trapping in postischemic hearts. Isolated rat hearts were subjected to 30 min of 37 degrees C, no-flow ischemia. Hearts were initially reperfused with diluted whole blood containing fluorescent leukocytes. At 5, (R5), 20, and 35 min of reperfusion, the deposition of leukocytes in the coronary capillaries and venules was observed directly using intravital fluorescence microscopy. Three groups were studied: a non-ischemic control group (group I) and postischemic groups reperfused with diluted whole blood treated with vehicle group II or PTX (5 mM; group III). Postischemic reperfusion with unactivated blood caused a significant trapping of leukocytes in coronary capillaries throughout reperfusion (R5, group I = 2.0 +/- 0.3 vs. group II = 5.7 +/- 0.6 leukocytes/capillary field, p < 0.05). The addition of PTX reduced capillary leukocyte trapping below control values throughout reperfusion (R5, group III = 1.6 +/- 0.2 leukocytes/capillary field, p < 0.05). At R5, there was no statistically significant difference in leukocyte accumulation in venules for all groups (group I = 1.5 +/- 0.6, group II = 3.2 +/- 0.4, group III = 3.3 +/- 0.4 leukocytes/100 microns venule). During the reperfusion period, leukocyte persistence in the capillaries of postischemic hearts (36%) was greater than in the venules (13%). These data indicate that early in reperfusion after myocardial ischemia, leukocyte trapping occurs primarily in the coronary capillaries. PTX reduced early leukocyte trapping in the capillaries. The results also demonstrate that during reperfusion, the mechanisms affecting capillary retention are more persistent than those in the venule. These findings suggest that attempts to attenuate the damaging potential of early leukostasis in capillaries consider the biophysical properties of the leukocyte.

Early in reperfusion following myocardial ischemia, leukocyte activation is necessary for venular adhesion but not capillary retention.

OBJECTIVE: The pathobiology of leukocyte sequestration in the coronary microcirculation following ischemia is unclear. We examined the location(s) and persistence of leukocyte sequestration of unactivated and preactivated blood in the coronary microcirculation early during reperfusion following ischemia. METHODS: Isolated rat hearts were subjected to 30 min of 37 degrees C, no-flow ischemia. Hearts were initially reperfused with diluted whole blood containing fluorescent leukocytes (DWB*). At 5, 20, and 35 min of reperfusion (R), the deposition of leukocytes in the coronary capillaries and venules was observed directly using intravital fluorescence microscopy. Four groups were studied: a nonischemic control group (Gr I), and postischemic groups reperfused with DWB* treated with vehicle (Gr II) or preactivated with 10(-8) M N-formylmethionyl-leucyl-phenylalanine (fMLP) (Gr III) or 10(-6) M fMLP (Gr IV). RESULTS: At R5, postischemic reperfusion with unactivated blood caused a significant trapping of leukocytes in coronary capillaries (Gr I = 2.2 +/- 0.4 versus Gr II = 5.6 +/- 0.6 leukocytes per capillary field, P < 0.05). Hearts in Gr IV exhibited significantly greater leukocyte retention in capillaries compared to all other groups at R5 (R5, Gr IV = 8.8 leukocytes per capillary field, P < 0.05) and at R35. At R5, although more leukocytes were observed adhered to the venules in Gr II compared to Gr I, the difference was not statistically significant (Gr I = 1.7 +/- 0.7 versus Gr II = 3.4 +/- 0.5 leukocytes per 100 microns venule, P = 0.23). DWB* preactivated with the lower concentration of fMLP (10(-8) M) resulted in a significant increase in venular leukocyte adhesion at R5 compared to Gr I and Gr II (Gr III 6.1 +/- 0.5, P < 0.05). After 35 min of reperfusion, a greater percentage of leukocytes remained in the capillaries than in the venules. CONCLUSIONS: These direct observations suggest that early in reperfusion after ischemia, both leukocyte and endothelial activation are necessary for venular adhesion, but that ischemia-induced coronary microvascular alterations are sufficient to promote leukocyte retention in coronary capillaries. These results also indicate that during 35 min of reperfusion, the degree of leukocyte washout is greater in the venules than in the capillaries. These results suggest that the mechanisms contributing to leukocyte retention early in reperfusion following myocardial ischemia are, indeed, different in the capillaries and venules and that the mechanisms affecting retention in capillaries are more persistent than those in the venules.

Left ventricular function after extended hypothermic preservation of the heart is dependent on functional coronary capillarity.

BACKGROUND: A growing body of knowledge has led to the hypothesis that injury to the microcirculation during hypothermic myocardial preservation may result in decreased contractility of hearts upon reperfusion. METHODS AND RESULTS: To test this hypothesis, we examined the relationship between no-reflow and left ventricular function after hypothermic cardiac preservation after reperfusion with solutions containing dilute whole blood (DWB) or washed red blood cells (K2RBC). Rat hearts were arrested with high-potassium cardioplegia, then flushed and stored for 6 hours in low-potassium cardioplegia at 4 degrees C. Hearts were reperfused at a constant flow rate (4 mL/min) with K2RBC for 60 minutes (group 1, n = 5) or DWB for 7 minutes followed by 53 minutes of K2RBC (group 2, n = 5). Left ventricular developed pressure (LVDP) was measured with an intraventricular balloon. Immediately after functional assessment, hearts were perfused with an india ink solution to mark flow, then glutaraldehyde. Morphometric techniques were used to determine the degree of capillary compression [delta d(c)], perfused capillary number per fiber area [QA(0)P], and perfused capillary surface area per fiber volume [Sv(c,f)P]. Capillaries were moderately compressed in both groups after reperfusion (group 1, 19 +/- 1%; group 2, 20 +/- 1%). QA(0)P and Sv(c,f)P were highly correlated with delta d(c) in hearts reperfused with K2RBC (r = .92 and r = .92; P < .01). Although statistically significant, the correlation was not as strong in DWB-reperfused hearts (r = .66 and r = .67; P < .05). LVDP was correlated to QA(0)P and Sv(c,f)P (r = .86 and r = .87, respectively) for groups 1 and 2. CONCLUSIONS: The weaker correlation between capillary perfusion and capillary compression in DWB-reperfused hearts suggests that factors other than compression contribute to no-reflow after hypothermic preservation. Regardless of the composition of the reperfusate, recovery of left ventricular function after hypothermic ischemia is directly related to coronary capillary perfusion upon reperfusion.

The initial response of blood leukocytes to incubation with perfluorocarbon blood substitute emulsions.

Due to their enhanced oxygen carrying characteristics, perfluorocarbon emulsions are valuable adjuncts to coronary revascularization procedures. However, the effects of perfluorocarbon emulsions on white blood cell function are unclear. The purpose of this study was to determine the effects of three perfluorocarbon emulsions on the adhesion characteristics of leukocytes. Heparinized blood from donor rats was mixed with either Phosphate Buffered Saline (PBS), PFB-1, PFB-2 (both non-complement activating perflubron emulsions, Alliance Pharmaceutical Corp.) or Fluosol (20% w/v PFC, Alpha Therapeutic) in a ratio of one part emulsion to six parts blood. The blood-emulsion mixtures were incubated for ten minutes at 37 degrees C, then passed down nylon wool leukocyte adhesion columns. Blood samples were counted for: Leukocytes, Granulocytes and Lymphocytes. % Cell Adherence was calculated as: (1-[WBC-out]/[WBC-in]). We found that, compared to PBS control, the granulocyte adherence to the nylon fiber was significantly increased for both Fluosol-20 and PFB-1 (P < 0.05). In contrast, PFB-2 (designed for reduced cell surface activity) caused no significant change in leukocyte adhesion. In fact, a modest decrease in granulocyte adhesion was observed with PFB-2. These findings suggest a direct effect of some, but not all, PFC emulsions on blood leukocyte function.

Brief perfusion with diluted whole blood after global myocardial ischaemia increases reperfusion injury.

OBJECTIVES: In vivo studies indicate that blood components, especially leucocytes, contribute to reperfusion injury after myocardial ischaemia. This study was designed to: (1) develop a small animal heart model of ischaemia-reperfusion that demonstrates the contribution of blood to reperfusion injury; (2) determine when the presence of blood in the heart--that is, during ischaemia or during early reperfusion--caused greater dysfunction; and (3) attempt to limit the blood contribution to reperfusion injury by leucocyte depletion. METHODS: Adult rat hearts were perfused in situ with a Krebs-albumin red cell solution (K2RBC), then isolated. Cardiac pump function was assessed with an intraventricular balloon as left ventricular developed pressure and contractility (dP/dt). Group I served as a non-ischaemic control group. Group II was subjected to global, no flow ischaemia for 30 min followed by 45 min reperfusion. In group III, diluted whole blood replaced the K2RBC for five min immediately before ischaemia. In group IV, diluted whole blood was perfused during the first five min reperfusion. In group V, the hearts were reperfused with leucocyte poor diluted whole blood. RESULTS: Pre-ischaemic pump function values were similar to other blood perfused, isolated heart models. Group I showed no increase in coronary resistance or decrease in pump function with time or in response to diluted whole blood. After 35 min reperfusion, the recovery (% control) of dP/dt in group II was 56(12), in group III it was 39(15) and in group IV it was only 19(6) (p < 0.05). Large increases in coronary vascular resistance, oedema, and contracture during reperfusion were also seen in group IV. When leucocytes were depleted from the diluted whole blood (group V), the recoveries were similar to reperfusion without diluted whole blood (group II). CONCLUSIONS: Thirty min of global, normothermic ischaemia caused significant cardiac dysfunction early during reperfusion. Perfusion with unstimulated blood for a limited period further impaired the recovery of function and enhanced myocardial oedema. Dysfunction was particularly evident when diluted whole blood was perfused during the first minutes of reperfusion. The leucocyte depletion studies suggest that leucocytes are necessary, but may not be sufficient, to demonstrate the blood contribution to reperfusion injury.

Microvascular compression during myocardial ischemia: mechanistic basis for no-reflow phenomenon.

Alterations in fiber size and capillary diameter were highly correlated with perfusion deficits after myocardial ischemia. After 5 (n = 3) and 30 (n = 5) min of global normothermic ischemia, isolated rabbit hearts were perfused with India ink and then with glutaraldehyde. Morphometric techniques were used to determine mean fiber cross-sectional area [a(f)], mean effective capillary diameter [d(c)], total and perfused capillary number per fiber area, and capillary length per fiber volume in subepicardium (Epi) and subendocardium (Endo). Sarcomere length was measured to differentiate between effects of fiber shortening and intracellular edema on a(f). After 30 min of ischemia, a(f) increased 41 (Epi) and 36% (Endo). Of these percentages, fiber shortening accounted for 2 (Epi) and 25% (Endo). Decreased d(c) was correlated with increased a(f) as well as reductions in perfused capillary number and length. Whereas intracellular edema had the greatest overall effect on a(f), fiber shortening accounted for a significant increase of a(f) in Endo, where perfusion deficits were most pronounced. These data support the hypothesis that microvascular compression consequent to increased a(f) contributes to perfusion deficits after myocardial ischemia.

Platelets do not modulate leukocyte-mediated coronary microvascular damage during early reperfusion.

Several studies indicate that leukocytes and platelets exacerbate the compromise of myocardial function that occurs after ischemia-reperfusion (I/R). However, it is unclear whether both leukocytes and platelets must be present to mediate coronary microvascular damage early during reperfusion after ischemia. To examine the effects of leukocytes and platelets on microvascular damage after I/R, we measured transcoronary albumin extravasation (O/I), perfused coronary capillary density (Caps), and transcoronary albumin extravasation per perfused capillary [(O/I)/Caps] in isolated rat hearts perfused with a Krebs-albumin-red blood cell solution [K(2)RBC], whole rat blood diluted with Krebs buffer (DWB), leukocyte-free, platelet-rich DWB (LFB), or leukocyte-rich, platelet-free DWB (LRB) before and after a 30-min period of global, no-flow ischemia. We found that in isolated hearts perfused with K(2)RBC before ischemia, O/I values were significantly increased (+68%, P < 0.01) and Caps values were significantly decreased (-25%, P < 0.05) after 25 min of reperfusion. A similar pattern of O/I values (+72%, P < 0.01) and Caps values (-40%, P < 0.05) was observed in hearts perfused with LFB. These effects were exacerbated in hearts perfused with DWB or LRB. O/I values were increased 90% (P < 0.01), and Caps values were decreased 62% (P < 0.01) in the DWB-perfused hearts. Similar increases in O/I values (+82%, P < 0.01) and decreases in Caps values (-65%, P < 0.01) were measured in the LRB-perfused hearts. Additionally, (O/I)/Caps values were significantly increased in the hearts perfused with DWB (+93%, P < 0.01) and LRB (+84%, P < 0.01) compared with the hearts perfused with K(2)RBC or LFB. These results suggest that interactions between leukocytes and platelets are not requisite for the development of coronary microvascular damage early during reperfusion after ischemia.

Stimulated leukocyte adhesion in coronary microcirculation is reduced by a calcium antagonist.

The first step in the acute myocardial inflammatory response is leukocyte sequestration in the coronary microcirculation. To determine the location(s) of stimulated leukocyte deposition in the coronary microcirculation and the effects of the calcium antagonist, nisoldipine, on leukocyte adhesion, leukocytes were stimulated with the chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine (FMLP) and blood cell adherence was evaluated using two methods. In vitro leukostasis was evaluated by measuring the extraction of white cells in nylon fiber columns. We found that diluted whole blood (DWB) demonstrated 30% granulocyte adherence. The chemotactic peptide FMLP (1 microM) significantly increased adherence to 69%. Pretreatment of the blood with nisoldipine (1 microM) immediately before FMLP significantly reduced the FMLP-induced adhesion to 47%. In the coronary microcirculation, FMLP caused a marked increase in leukocyte sequestration, primarily in coronary capillaries. The FMLP effect was somewhat transient because the washout of trapped white cells was similar in the vehicle and FMLP groups. Nisoldipine significantly reduced the FMLP-induced leukostasis in coronary capillaries (P < 0.05). The magnitude of the attenuation of leukostasis with nisoldipine was remarkably similar in both models, suggesting a direct effect of this agent on the blood rather than on the blood vessels. These findings offer another possible mechanism by which dihydropyridine calcium antagonists may be cardioprotective under pathophysiological conditions.

Spatial relationship between leukocyte accumulation and microvascular injury during reperfusion following hepatic ischemia.

In order to further elucidate the possible contribution of leukocytes to microvascular injury during reperfusion following total hepatic ischemia, we studied the spatial relationship between areas of white cell accumulation and areas of microvascular damage in the rat liver in vivo. No-flow hepatic ischemia was produced for 90 min in vivo and during the ensuing reperfusion phase (I/R) leukocyte accumulation, absolute number of perfused sinusoids per unit area, and red blood cell velocity were quantitated using in vivo epi-fluorescence video microscopy. The total number of stationary leukocytes in the liver during reperfusion was found to be significantly elevated following ischemia compared to time-matched sham-operated controls. In addition, by 2 hr of reperfusion, approximately 80% of the leukocytes in the I/R group were extravascular compared to only about 50% in the controls. When leukocyte accumulation and microhemodynamics were expressed on the basis of whole liver, the increased accumulation of leukocytes was associated with decreased microvascular perfusion as indicated by decreased number of sinusoids perfused and decreased red blood cell velocity. However, when the data were analyzed on the basis of .05mm2 microscopic fields on the surface of the liver, there was no difference in leukocyte accumulation in areas with sinusoidal blood flow compared to areas that were devoid of perfused sinusoids. Moreover, in a correlation analysis of number of adherent leukocytes/microscopic field vs red blood cell velocity in perfused sinusoids in that field, only a very small negative correlation between leukocytes/field and red blood cell velocity was found (r = -.23, p < .05). These results demonstrate that at the whole organ level leukocyte accumulation appears to correlate well with microvascular damage; however, this increase in whole liver accumulation of leukocytes does not necessarily reflect accumulation at sites of overt microvascular damage. Thus, leukocyte-independent factors are likely to be of considerable quantitative importance in microvascular injury during reperfusion following hepatic ischemia.

The microvascular pathophysiology of chronic venous insufficiency.

Severe chronic venous insufficiency (CVI) demonstrates as chronic, hard-to-heal wounds of the lower extremity. The wound is the result of poor skin perfusion due to a complex series of pathologic events, often initiated by a deep vein thrombosis (DVT). As years pass, the DVT causes venous valvular damage and incompetence. The calf muscle pump fails to augment venous return, and venous blood pressure is chronically elevated upon standing. Mechanisms that normally prevent the transmission of venous hypertension back upstream to the dermal microcirculation are lost. Early dermal microvascular responses include increased fluid filtration and edema. An inflammatory response induces white cell activation and adhesion. It is thought that activated white cells are trapped in dermal capillaries and increase microvascular permeability. Plasma proteins leak into the tissue space, increasing the edema. Ischemic damage to the epidermis leads to epithelial cell necrosis and ulceration. The ulcer is often slow to heal, due to inadequate perfusion and delivery of substrates required for proper wound healing. Current treatments aim to improve calf pump function, reduce edema, improve perfusion, and enhance wound healing.

Early in reperfusion, leukocytes alter perfused coronary capillarity and vascular resistance.

Myocardial no-reflow is a critical consequence of myocardial ischemia-reperfusion (I/R). Recent studies indicate that formed blood elements (e.g., leukocytes and platelets) contribute greatly to the compromise of myocardial blood flow that occurs after I/R. To assess the contributions of leukocytes and platelets to alterations in microvascular perfusion, we measured total coronary vascular resistance and perfused coronary capillary density before and after a 30-min period of no-flow ischemia in isolated rat hearts perfused with either 1) a Krebs-albumin-red cell solution [K(2)RBC]; 2) diluted whole blood (DWB) with Krebs (1:1); or 3) leukocyte-free DWB (LFB). We found that hearts perfused with K(2)RBC before ischemia demonstrated a significant decrease in perfused capillarity (-25%, P less than 0.05) after 25 min of reperfusion. Hearts perfused with LFB before ischemia exhibited a similar decrease in perfused capillarity (-33%, P less than 0.05) during reperfusion. However, in the DWB-perfused hearts, there was a 62% decrease in perfused capillarity (-62%, P less than 0.01) during reperfusion. Moreover, during reperfusion, total coronary vascular resistance was elevated significantly (+76%, P less than 0.01) in the DWB-perfused hearts but not in either the K(2)RBC or LFB groups. These results indicate that 1) platelets do not play a major role in alterations of microvascular perfusion after ischemia; 2) leukocytes are not requisite for the development of microvascular no-reflow early in reperfusion but their presence further exacerbates this deleterious effect; and 3) a relationship exists between perfused capillarity and vascular resistance in the isolated rat heart after global ischemia.