Paul F. McDonagh

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.

Thrombin activity and platelet microparticle formation are increased in type 2 diabetic platelets: a potential correlation with caspase activation.

Diabetics suffer from many complications including a prothrombotic condition. Activated platelet membrane provides an anchor, phosphatidylserine, for the attachment of the prothrombinase complex, which allows increased thrombin formation. This study aimed to further elucidate the interrelationship between coagulation proteins and activated platelets in type 2 diabetic blood. We found that there was a significant increase (30 x) in thrombin activity in the type 2 diabetic (ZDF) blood as compared to age-matched (ZL) controls (p<0.001). There was also a significant increase in the number of platelet microparticles in the type 2 diabetic rat compared to the lean control (p<0.001). Further, there were significant increases in caspase-3, -6, and -8 activities in the type 2 diabetic rats as compared to the lean controls (p<0.05). The combination of increased thrombin activity, increased PMP formation and increased caspase activity may contribute to the hypercoagulability of the diabetic blood. These results give more insight into the mechanisms underlying the interrelationship between diabetic platelets and coagulation proteins causing a prothrombotic condition in this patient population at increased risk from thromboembolic events.

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.

Proinflammatory cytokines are increased in type 2 diabetic women with cardiovascular disease

Diabetics have a much greater morbidity and mortality due to cardiovascular disease (CVD) than nondiabetics. Furthermore, diabetic women have a 3.8-fold greater risk for CVD compared to diabetic men. Inflammation is now considered a risk factor for CVD and it has been demonstrated that inflammation also plays a role in diabetes. One component of inflammation that has reported to be increased in patients with diabetes only and CVD only are proinflammatory cytokines, particularly interleukin-6 (IL-6), tumor necrosis factor (TNF-alpha), and interleukin-1 (IL-1beta). This study was performed to test the hypothesis that these proinflammatory cytokines were increased in women with CVD and further increased in diabetic women with CVD compared to nondiabetic women with CVD and healthy age-matched controls. We found that IL-6 was increased in diabetic women with CVD compared to healthy age-matched controls (1.41 = 0.48 to 0.33 +/- 0.06 pg/ml, P < .05). IL-6 was also increased in diabetic women without CVD compared to healthy age-matched controls, but not significantly (0.96 +/- 0.27 to 0.33 +/- 0.06 pg/ml). We found that TNF-alpha was increased in diabetic women with and without CVD compared to healthy age-matched controls, but not significantly (4.53 +/- 1.38 to 3.93 +/ -0.53 to 2.33 +/- 0.89 pg/ml). IL-1beta was not significantly different among any of the four groups of women. These results indicate that both IL-6 and TNF-alpha are chronically increased in diabetic women with and without CVD compared to nondiabetic women. The additive concentration of cytokines in diabetes and CVD suggests a common inflammatory state in both diabetes and CVD.

Microvascular perfusion and transport in the diabetic heart.

Diabetes is a chronic disease of metabolic dysfunction that is increasing worldwide. 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 changes 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.

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.

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-μm 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.

Protective effects of inhibiting both blood and vascular selectins after stroke and reperfusion.

Abstract Early intervention after acute ischemic stroke is essential to minimize brain cell injury. Although reperfusion of the ischemic brain is the treatment of choice for acute stroke, reperfusion itself may cause additional injury. The inflammatory cascade, characterized in part by early leukocyte interaction with endothelium, may contribute to this additional injury to blood vessels and surrounding brain tissue, extending the area of infarction. The selectin family of adhesion molecules mediates the initial, rolling and tethering of leukocytes to endothelium. P-selectin is rapidly expressed on ischemic endothelium in the brain vasculature, and L-selectin is expressed on leukocytes. Blocking the selectin-mediated tethering step may limit the inflammatory component of reperfusion injury in the brain. Fucoidin (FCN), a competitive inhibitor of P- and L-selectin, has been reported to decrease leukocyte accumulation during reperfusion of other organs. The effect of both leukocyte and endothelial selectin inhibition after cerebral ischemia and reperfusion has not been previously examined. The purpose of this study was to determine the effects of selectin adhesion molecule blockade on cerebral infarction size and neurological function after middle cerebral artery occlusion and reperfusion (MCAO-R) in the rat. MCAO was induced using the filament method. All animals were subjected to 4 h of MCAO and 24 h of reperfusion. After 24 h, brains were analyzed for size of infarction. Neurological function was assessed during stroke and 24 h after reperfusion. Two groups were studied, an untreated control group (n = 9) and a group treated with the selectin inhibitor, fucoidin (25 mg kg-1) (n = 9). We found that selectin blockade significantly reduced cerebral infarction size by 50% (p < 0.05) and improved neurological function (p < 0.05). In addition, a trend toward decreased cerebral edema was demonstrated with selectin inhibition. These results indicate that treatment of the blood and the endothelium with a selectin anti-inflammatory agent is protective after focal stroke and reperfusion in the rat. [Neurol Res 2002; 24: 226-232]

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 microcircu‐lation 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.

Complement inhibition reduces injury in the type 2 diabetic heart following ischemia and reperfusion

Chronic inflammation exacerbates the cardiovascular complications of diabetes. Complement activation plays an important role in the inflammatory response and is known to be involved in ischemia-reperfusion (I/R) injury in the nondiabetic heart. The purpose of this study was to determine if increased complement deposition explains, in part, the increased severity of neutrophil-mediated I/R injury in the type 2 diabetic heart. Nondiabetic Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats underwent 30 min of coronary artery occlusion followed by 120 min of reperfusion. Another group of ZDF rats was treated with the complement inhibitor FUT-175 before reperfusion. Left ventricular (LV) tissue samples were stained for complement deposition and neutrophil accumulation following reperfusion. We found significantly more complement deposition in the ZDF LV compared with the ZLC (P < 0.05), and complement deposition was associated with significantly greater neutrophil accumulation. In whole blood samples taken preischemia and at 120 min reperfusion, neutrophils exhibited significantly more CD11b expression in the ZDF group compared with the ZLC group (P < 0.05). Furthermore, intracellular adhesion molecule (ICAM)-1 expression following I/R was increased significantly in ZDF hearts compared with ZLC hearts (P < 0.001). These results indicate that, in the ZDF heart, increased ICAM-1 and polymorphonuclear neutrophil (PMN) CD11b expression play a role in increasing PMN accumulation following I/R. The infarct size of the ZDF was significantly greater than ZLC (P < 0.05), and treatment with FUT-175 significantly decreased infarct size, complement deposition, and PMN accumulation in the diabetic heart. These findings indicate an exacerbated inflammatory response in the type 2 diabetic heart that contributes to the increased tissue injury observed following ischemia and reperfusion.