Decrease In Functional Capillary Density Research Articles

Pathophysiological mechanisms of chronic venous disease and their role in C0s clinical class

The aim of this review is to provide a clear understanding of the pathophysiological mechanisms of chronic venous disease (CVD) at different clinical stages and the possible role of these mechanisms in the development of symptoms in C0s clinical class of the Clinical, Etiologic, Anatomic, and Pathophysiologic classification, which consists of symptomatic patients without any visible or palpable signs of venous disease. The prevalence of C0s class in several epidemiological studies varies between 13% and 23% of the general population. Wall remodeling and valve destruction due to white cell endothelial interaction is the main cause of primary varicose veins, while deep vein thrombosis produces secondary changes leading to the postthrombotic syndrome. The underlying mechanism of the skin changes and ulceration is venous hypertension, which is transmitted to the skin microcirculation. Over the last 10 years, an improved videocapillaroscopic technique, the orthogonal polarization spectral imaging technique demonstrated that quantitative measurements in the skin microcirculation are progressively altered from C1 to C6 patients and that values in CVD patients are significantly different from healthy individuals (P < 0.05): capillary diameter increases and capillary morphology worsens from C2 to C5; diameter of the dermal papilla and diameter of the capillary bulk increase from C3 to C5; and functional capillary density (FCD) decreases from C4 to C5. In addition, significant changes have been shown between C0a and C0s patients despite the presence of normal conventional duplex scans in the latter: a decrease of FCD and an increase in the diameter of the dermal papilla. Functional abnormalities found to be present in C0s patients by recent studies include increased compliance of the venous wall (hypotonic phlebopathy), dilatation of deep veins in the calf producing an abnormally increased venous volume, reduction in emptying of venous reservoir, reduction in the venoarteriolar response on standing, and blood reflux in small venules despite a normal conventional duplex scan. However, most of the studies are small, and their findings need to be confirmed by larger series. It remains to be seen whether functional changes and microcirculatory changes respond to venoactive medications in parallel to the relief of symptoms.

142 Intravital imaging of leukocyte, platelet and stem cell trafficking in vivo in the cardiac microcirculation following myocardial ischaemia-reperfusion injury

BackgroundThe clinical success of stem cell (SC) therapy for myocardial infarction is compromised by poor cardiac homing following systemic delivery. As such therapy may depend on beneficial paracrine effects, it...

Reduced uterine perfusion pressure decreases functional capillary density in skeletal muscle.

The purpose of this study was to examine the functional and structural capillary density in the reduced uterine perfusion pressure (RUPP) model, which when performed during pregnancy is an established animal model of preeclampsia. We hypothesized that the RUPP model would be associated with capillary rarefaction and impaired capillary perfusion, which would be more pronounced in the pregnant state. Female Sprague-Dawley rats (n = 32) were randomized to nonpregnancy (Nonpregnant) or breeding (Pregnant) at 12 wk of age and again to RUPP or SHAM surgeries on gestational day (GD) 14 (or equivalent age in nonpregnant rats). On GD 20 (or equivalent), capillary structure and perfusion of the extensor digitorum longus were imaged using digital intravital video microscopy. Functional videos were analyzed by a blinded observer to measure capillary density, expressed as capillaries per millimeter intersecting three staggered reference lines (200 μm). Flow was scored as the percentage of capillaries having 1) continuous, 2) intermittent, or 3) stopped flow. Total capillary density was not different between groups. There was a main effect of RUPP surgery resulting in decreased continuous flow vessels (P < 0.01) and increased stopped flow (P < 0.01), which was driven by differences between pregnant animals (Continuous flow: pregnant SHAM 80.1 ± 7.8% vs. pregnant RUPP 67.8 ± 11.2%, P < 0.05) (Stopped flow: pregnant SHAM 8.7 ± 3.2% vs. pregnant RUPP 17.9 ± 5.7%, P < 0.01). Our results demonstrate that the RUPP surgery is associated with a decrease in functional capillary density in skeletal muscle that is more pronounced in the pregnant state, which may contribute to the vascular pathophysiology observed in preeclampsia.

Statins Decrease Neuroinflammation and Prevent Cognitive Impairment after Cerebral Malaria

Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.

NUTRITIVE PERFUSION OF PANCREATIC ENDOCRINE TISSUE DURING HEMORRHAGIC HYPOTENSION

Although the exocrine gland frequently has I/R-associated complications such as posttransplant pancreatitis, hypoxia-induced dysfunction of pancreatic endocrine tissue is rarely observed. However, sympathetic hypersensitivity is accused of impaired endocrine function observed in human pancreatic grafts. These tissue-confined differences in susceptibility might be attributed to a distinct islet-specific regulatory control of blood flow (BF). To investigate this hypothesis, intravital microscopy was used for visualization of islets in situ and revascularized islet isografts in Syrian golden hamsters. Blood withdrawal was performed to induce sympathetic stimulation and to evoke an appropriate stress response of the tissue under investigation. Hypotension resulted in a perfusion pressure-dependent reduction of perfusion in both islets in situ and islet isografts. This was associated with comparable microhemodynamics of the tissues in direct vicinity, that is, pancreatic exocrine and host muscle tissue. There was a progressive decrease in functional capillary density of islets in situ and islet isografts that significantly correlated with the stepwise reduction of arterial blood pressure and did not differ in the hypotension-induced perfusion pattern in the neighboring exocrine and host skeletal muscle tissue. Concomitantly, capillary BF in islets in situ and islet isografts and in pancreatic exocrine and host muscle tissue was found to be reduced due to hypotension-associated decreases in capillary diameters and BF velocity. Microvascular perfusion of pancreatic islets and islet isografts is not preserved but strongly parallels perfusion pattern of the neighboring tissue under hemorrhagic stress. This disproves the existence of an individual islet-specific regulatory control of blood flow.

Pancreatic Ischemia/Reperfusion Injury

Pancreatic ischemia/reperfusion injury (IRI) can influence the results after transplantation. Temperature during ischemia can affect IRI. A temperature of 4 degrees C is assumed as optimal for graft preservation. There are no data about the impact of different ischemia temperatures in pancreatic IRI. Ischemia/reperfusion injury was induced in pancreatic tail segments (2-hour ischemia, 2-hour reperfusion), with rats (7/group) without ischemia served as control. Animals were randomized to the different experimental groups. To achieve the desired temperature (4, 18, or 37 degrees C and 37 degrees C control), pancreatic tail segments were superfused with temperated saline. After reperfusion, microcirculation was observed by intravital fluorescence microscopy. Functional capillary density (FCD), leukocyte adherence in post-capillary venules, and histological damage were analyzed. In IRI groups, decrease of FCD 1 and 2 hours after reperfusion compared with baseline measurements was significant. Functional capillary density in 4 degrees C was better as compared with 18 and 37 degrees C after reperfusion. Lower adherent leukocytes were seen in 4 and 18 degrees C, compared with 37 degrees C and also to CO. In 4 degrees C, histological damage was lower as compared with 18 and 37 degrees C. We could demonstrate that also in pancreatic IRI, tissue injury is temperature dependent. Compared with 37 degrees C, although a protective effect is established already at 18 degrees C, more protection is achieved with storage at 4 degrees C. Our data suggest that 4 degrees C has the best protective effect on pancreatic IRI.

Ischemia-Induced Up-Regulation of Heme Oxygenase-1 Protects From Apoptotic Cell Death and Tissue Necrosis

Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Selective blockade of endothelin-B receptor improves survival of critically perfused musculocutaneous flaps

Insufficient perfusion of distal flap areas, which may lead to partial necrosis, still represents a challenge in reconstructive surgery. In the process of microvascular and endothelial dysfunction, endothelins (ETs) and their receptors may play an important role. Therefore, the aim of the study was to investigate in a chronic in vivo model the effect of various ET-receptor antagonists in critically perfused flap tissue. A random pattern musculocutaneous flap was elevated in the back of 25 C57BL/6 mice and fixed into a dorsal skinfold chamber. Repetitive intravital fluorescence microscopy was performed over a 10-day observation period, assessing arteriolar diameter, arteriolar blood flow (aBF), functional capillary density (FCD), the area of tissue necrosis, and the development of newly formed blood vessels. ET-receptor blockers were administrated intraperitoneally 30 min before induction of ischemia, as well as daily during the subsequent 4-day period, including (1) BQ-123, a specific ET-A-receptor antagonist (ET-A = 1 mg/kg), (2) BQ-788, a selective ET-B-receptor antagonist (ET-B = 1 mg/kg), and (3) PD-142893, a nonselective ET-AB-receptor antagonist (ET-AB = 0.5 mg/kg). Animals receiving saline only served as controls (n = 7). Despite an increase in aBF during the 10-day observation period (day 1 = 1.92 +/- 0.29 nl/s; day 10 = 4.70 +/- 1.64 nl/s), the flaps of saline-treated controls showed a distinct decrease in FCD (94 +/- 12 cm/cm(2)). This perfusion failure resulted in flap necrosis of 52 +/- 3%. Selective blockade of the ET-B receptor caused a further increase in aBF already at day 1 (2.97 +/- 0.42 nl/s), which persisted during the following 10-day observation period (day 10 = 5.74 +/- 0.69 nl/s). Accordingly, adequate FCD could be maintained (day 10 = 215 +/- 8 cm/cm(2); p < 0.05 vs control), resulting in a significant reduction in flap necrosis (day 10 = 25 +/- 4%; p < 0,05). In contrast, neither selective blockade of the ET-A receptor nor nonselective ET-A- and ET-B-receptor blockade were able to significantly affect aBF when compared to controls (day 1 = ET-A = 1.39 +/- 0.10 nl/s; ET-AB = 1.53 +/- 0.80 nl/s; n.s.). Accordingly, flap necrosis after ET-A- and ET-AB-receptor inhibition did not differ from that of controls (day 10 = ET-A: 46 +/- 10%; ET-AB = 51 +/- 7%). Our data show that only selective ET-B-receptor inhibition is capable of maintaining nutritive perfusion and, hence, reducing necrosis in critically perfused flap tissue. Accordingly, administration of ET-B-receptor antagonists may be considered in the treatment of critically perfused flaps.

Microvascular and capillary perfusion following glycocalyx degradation

Systemic parameters and microvascular and capillary hemodynamics were studied in the hamster window chamber model before and after hyaluronan degradation by intravenous injection of Streptomyces hyaluronidase (100 units, 40-50 U/ml plasma). Glycocalyx permeation was estimated using fluorescent markers of different molecular size (40, 70, and 2,000 kDa), and electrical charge. Systemic parameters (blood pressure, heart rate, blood gases) and microhemodynamics (vascular tone, velocity, and blood flow) remained statistically unchanged after injection of hyaluronidase, compared with inactivated hyaluronidase. Conversely, capillary hemodynamics were drastically affected. Functional capillary density, the capillaries perfused with red blood cells (RBCs), decreased by 35%, capillary Hct of the remaining functional capillaries increased from 16 to 27%, and penetration of 70-kDa fluorescent marker increased. Furthermore, plasma-only perfused capillaries statistically increased 30 min after hyaluronidase. The decrease in functional capillary density accounted for an increased RBC flux in the remainder of the capillaries, since the same number of RBCs had to traverse a reduced number of capillaries. Flux balances showed a reduction from baseline of 11% for the RBC flux and 20% for the plasma flux after treatment. These discrepancies are within the margin of error of the techniques used and could be explained by accounting for RBC over-velocity compared with plasma. These findings suggest that the decrease in the glycocalyx leads to capillary perfusion impairments.

Local Cooling Restores Microcirculatory Hemodynamics after Closed Soft-Tissue Trauma in Rats

Severe closed soft-tissue injury (CSTI) results in progressively developing microvascular dysfunction and local inflammation. Cooling reduces swelling, pain, cellular oxygen demand, and metabolic activity. However, effects of cooling on posttraumatic microcirculation are not yet fully understood. Thus, we assessed effects of local cooling on microcirculation, regional inflammatory response including leukocyte-endothelial cell interaction, and edema formation after CSTI. Standardized CSTI was induced by means of controlled impact injury in the left tibial compartment of 14 male Sprague-Dawley rats. Rats were assigned to four groups (n = 7 per group) as follows: group I, no trauma/no cooling; group II, no trauma/20 minutes of cooling; group III, 1.5 hours posttrauma/no cooling; and group IV, 1.5 hours posttrauma/20 minutes of cooling. CSTI resulted in a significant decrease in functional capillary density, a marked increase in microvascular permeability, and granulocyte infiltration (HIS48) as revealed by intravital microscopy and immunohistochemistry of the left extensor digitorum longus muscle. After 20 minutes of local cooling, these microvascular derangements were restored to the level of controls (group I). Edema (extensor digitorum longus muscle wet-to-dry weight ratio) was less pronounced compared with noncooling conditions (group III). Immunoreactivity for HIS48 (neutrophilic granulocytes) in injured rats subjected to local cooling (group IV) was markedly decreased compared with noncooling conditions (group III). These results provide in vivo evidence that cooling affords protection of posttraumatic microcirculation through sustained inhibition of microvascular and endothelial dysfunction leading to less granulocyte-dependent inflammation and skeletal muscle edema. Local cooling appears to reduce propagation of acute microvascular injury, preventing leukocyte-dependent tissue destruction and escalation of secondary tissue damage after musculoskeletal soft-tissue trauma.

411 Microcirculatory Parameters as Early Markers for Infection in Preterm Infants

Background We have previously shown that orthogonal spectral imaging (OPS imaging) can be used to measure microcirculatory parameters such as Red Blood Cell Velocity (RBC Vel) and Functional Capillary Density (FCD). As neonatal infections often manifest unspecificly in e. g. paleness and prolonged capillary refill time we examined if a decrease in FCD can predict neonatal infection.Methods in 25 preterm infants (gestational age of 28 (26,18 - 27,58) wks; birth weight 900 (807,9 - 992,8) g) RBC Vel and FCD was determined from d 3 till d 30 of life. OPS imaging was applied to the upper arm near the axilla, images were stored on videotape and analyzed off-line with the CapiScope Program.Results There were 17 episodes of proven infections (PosInf) with either increased C-reactive Protein or increased interleukine-6. The day when antibiotic treatment was started was defined as d 0 of infection and the evaluation was concentrated on five days before (d -5 until d -1) and five days after (d +1 until d +5). In nine cases the suspected infection was not confirmed biochemically (NegInf). Four infants had no episode of infection for entire time. In PosInf we found a statistical significant decrease in FCD from d -5 to d -1 (mean (95% CI) 231 (187 - 236) cm/cm2 versus 234 (190 - 257) cm/cm2 ; p = 0,0127), but not for NegInf ( p = 0,58). RBC Vel did not change from d -5 to d -1 nor during infection.Conclusion A decreasing FCD correctly identified episodes of infections already one day earlier than changes in laboratory parameters. OPS-Imaging could be used for early diagnosis of secondary infection in preterm infants. However, these changes in FCD during infection are not represented by absolute values, but must be identified by daily intra-individually.

Increased leukocyte transit times through capillaries contributes to maldistribution of flow

Sepsis has been shown to result in a decrease in functional capillary density (FCD) in animal models and in septic patients. We have previously shown that increased numbers of stopped flow capillaries result in a fall in capillary oxygen saturation [1,2]. The goal of this study was to determine whether leukocytes played a role in capillary occlusion. Our objective was to examine leukocyte traffic within the microcirculation in the extensor digitorum longus (EDL) muscle using fluorescently (rhodamine 6G) labeled leukocytes and to correlate this with the loss of FCD seen in a 5 hour rat cecal ligation and perforation (CLP) model of sepsis. Ten rats were randomized to sham (n = 5) or CLP (n = 5). The capillary bed in the EDL muscle was observed using intravital video microscopy. Functional capillary density was determined prior to the loss of FCD at 1.5 hours after induction of sepsis or the sham procedure. The location of all capillaries within a single capillary network from arteriole to venule was mapped. At 4 hours rhodamine 6G was injected and the same capillary network was observed using fluorescence microscopy to determine which capillaries contained flowing or stopped leukocytes and to measure leukocyte transit times. Leukocytes were found to preferentially traverse a subpopulation of capillaries in both sham (40% of total capillary paths) and CLP animals (60%). In the sham animals, 80% of the leukocytes traversed the capillary bed in 30 s). In CLP animals 45 ± 5% of capillaries had stopped flowing by 5 hours; 63 ± 2% of these capillaries were preferential flow paths for leukocytes. The slowing of leukocytes through the capillary bed suggested the possible role of selectins. Six CLP animals were treated with fucoidin, a nonselective general inhibitor of selectins. Fucoidin prevented the increase in leukocyte transit times and the loss of FCD at 5 hours, indicating that selectins were involved in the slowing of leukocytes in sepsis. Leukocytes play a significant role in the maldistribution of capillary blood flow through the loss of perfused capillaries and increased leukocyte transit times. Both effects probably cause a concomitant fall in oxygen saturation in neighboring capillaries, leading to periods of tissue hypoxia.

Ischemic preconditioning fails to improve microcirculation but increases apoptotic cell death in experimental pancreas transplantation

Abstract Brief periods of warm ischemia and subsequent short reperfusion before either long-term cold or warm ischemic insult (ischemic preconditioning, IPC) have proven to ameliorate ischemia/reperfusion (I/R) injury in various organs, such as the liver and lung. The aim of this study was to examine the effect of IPC on pancreatic cell apoptosis and microcirculatory impairments in experimental pancreas transplantation. Male Lewis rats served as donors and recipients of heterotopic syngeneic pancreaticoduodenal transplantation. Recipient animals were divided into two experimental groups: group Tx (n= 7) received grafts without IPC, group Tx&IPC received grafts with IPC. Animals that had not undergone transplantation but whose pancreata had been exteriorized served as controls (n= 5). All pancreatic grafts were preserved in University of Wisconsin solution for 6 h at 4°C. IPC was induced by interruption of the arterial blood flow for 10 min followed by 10 min of reperfusion. One and two hours after reperfusion, graft microcirculation was assessed by means of intravital microscopy (IVM). Rats were immediately killed after the second measurement and DNA breaks of acinar cells were detected by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end-labelling (TUNEL) assay and gel electrophoresis (laddering). The apoptotic index (AI) was defined as the number of apoptotic cells per high-power field. Analysis of both groups of transplanted grafts showed a significant decrease in functional capillary density (FCD) and a significant increase in leukocyte sticking to postcapillary venules (LAV) at 1 h and 2 h of reperfusion, compared with animals that had not undergone transplantation (P<0.01). In parallel, AI was significantly increased in transplanted grafts compared to the controls (P<0.01). Grafts subjected to IPC showed no significant differences, neither for FCD nor LAV, at both time points if compared with grafts of group Tx. However, IPC resulted in a significant increase in AI (P<0.05). We can conclude that IPC has no effect on pancreatic microcirculation but enhances acinar cell apoptosis in experimental pancreas transplantation. These results indicate that IPC might increase I/R injury after pancreatic cold ischemia.

Ischemic preconditioning fails to improve microcirculation but increases apoptotic cell death in experimental pancreas transplantation.

Brief periods of warm ischemia and subsequent short reperfusion before either long-term cold or warm ischemic insult (ischemic preconditioning, IPC) have proven to ameliorate ischemia/reperfusion (I/R) injury in various organs, such as the liver and lung. The aim of this study was to examine the effect of IPC on pancreatic cell apoptosis and microcirculatory impairments in experimental pancreas transplantation. Male Lewis rats served as donors and recipients of heterotopic syngeneic pancreaticoduodenal transplantation. Recipient animals were divided into two experimental groups: group Tx (n=7) received grafts without IPC, group Tx&IPC received grafts with IPC. Animals that had not undergone transplantation but whose pancreata had been exteriorized served as controls (n=5). All pancreatic grafts were preserved in University of Wisconsin solution for 6 h at 4 degrees C. IPC was induced by interruption of the arterial blood flow for 10 min followed by 10 min of reperfusion. One and two hours after reperfusion, graft microcirculation was assessed by means of intravital microscopy (IVM). Rats were immediately killed after the second measurement and DNA breaks of acinar cells were detected by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end-labelling (TUNEL) assay and gel electrophoresis (laddering). The apoptotic index (AI) was defined as the number of apoptotic cells per high-power field. Analysis of both groups of transplanted grafts showed a significant decrease in functional capillary density (FCD) and a significant increase in leukocyte sticking to postcapillary venules (LAV) at 1 h and 2 h of reperfusion, compared with animals that had not undergone transplantation ( P<0.01). In parallel, AI was significantly increased in transplanted grafts compared to the controls ( P<0.01). Grafts subjected to IPC showed no significant differences, neither for FCD nor LAV, at both time points if compared with grafts of group Tx. However, IPC resulted in a significant increase in AI ( P<0.05). We can conclude that IPC has no effect on pancreatic microcirculation but enhances acinar cell apoptosis in experimental pancreas transplantation. These results indicate that IPC might increase I/R injury after pancreatic cold ischemia.

Endotoxin preconditioning in pancreatic ischemia/reperfusion injury.

Prospective organ donors are exposed to various stress types. The effect of endotoxin pretreatment (ETX) on pancreatic ischemia/reperfusion injury (IRI) is unclear. We investigated, using a rat model of pancreatic IRI of an in situ isolated pancreatic tail segment, the effect of ETX on postischemic microcirculation and organ damage. Twenty-four hours before pancreatic dissection, either intraperitoneal application of ETX (1 mg/kg in 0.9% NaCl) or saline only (control) was performed. Two-hour normothermic ischemia of the pancreatic tail was induced by clamping the splenic vessels and was followed by a reperfusion period of 2 hours. Microcirculatory parameters were measured by intravital epifluorescence microscopy [functional capillary density (FCD), adherent leukocytes (ALs), and histology]. The presented data represent the mean +/- SEM/SD as appropriate. ETX pretreatment caused a significantly greater decrease in FCD (497 +/- 6 cm/cm2 baseline versus 326 +/- 15 cm/cm2 2 hours of reperfusion) compared with controls (498 +/- 8 versus 258 +/- 15 cm/cm2) 2 hours after reperfusion (P < 0.01). Two hours after reperfusion, ALs were significantly decreased in ETX animals compared with controls (ETX: 141 +/- 37 versus 273 +/- 36 cells/mm2, P < 0.05). Histologic damage was less in ETX (6.4 score points +/- 0.32 versus 8.8 +/- 0.33 control, P < 0.05). ETX preconditioning decreases microcirculatory deterioration caused by IRI by means of less loss of nutritive tissue perfusion, decrease in ALs, and less histologic damage. This indicates a protective effect of ETX preconditioning in pancreatic IRI.

Endothelin-a receptor antagonist treatment improves the periosteal microcirculation after hindlimb ischemia and reperfusion in the rat.

To examine the microcirculatory changes in the rat tibial periosteum after hindlimb ischemia and reperfusion and to evaluate the effects of endothelin-A (ET-A) receptor antagonist therapy in this condition. The healing and functioning of vascularized bone autografts depend mainly on the patency of the microcirculation, and the activation of ET-A receptors may be an important component of the tissue response that occurs during ischemia-reoxygenation injuries. Wistar rats were subjected to 1 hour of hindlimb ischemia and 3 hours of reperfusion. The periosteal microcirculation was visualized by intravital fluorescence microscopy. The leukocyte rolling and adherence in the postcapillary venules and the functional capillary density of the periosteum were determined. Two separate groups were treated with the selective ET-A receptor antagonist BQ 610 or the novel ET-A receptor antagonist ETR-p1/fl peptide at the onset of reperfusion. Reperfusion was accompanied by a significant decrease in functional capillary density and by an increase in the primary and secondary leukocyte-endothelial cell interactions. ET-A receptor inhibition reduced the leukocyte rolling and firm adherence and attenuated the decrease in functional capillary density in both treated groups. ET-1 plays a major role in microvascular dysfunction in the periosteum during reperfusion. The ET-1-ET-A receptor system might be an important target for tissue salvage therapy in transplantation surgery.

Hydroxyethyl starch (130 kD), but not crystalloid volume support, improves microcirculation during normotensive endotoxemia.

Increased leukocyte-endothelial cell interaction (LE) and deterioration of capillary perfusion represent key mechanisms of septic organ dysfunction. The type of volume support, however, which may be used during septic disorders, remains controversial. Using intravital microscopy, the authors studied the effect of different regimens of clinically relevant volume support on endotoxin-induced microcirculatory disorders, including the synthetic colloid hydroxyethyl starch (HES, 130 kD) and a crystalloid regimen with isotonic saline solution (NaCl). In Syrian Golden hamsters, normotensive endotoxemia was induced by intravenous application of Escherichia coli lipopolysaccharide (LPS, 2 mg/kg). The microcirculation was analyzed in striated muscle of skinfold preparations. HES 130 kD (Voluven(R), 16 ml/kg, n = 7) or isotonic saline (NaCl, 66 ml/kg, n = 6) were infused 3 h after LPS exposure over a 1-h period (posttreatment mode). Animals receiving LPS without volume therapy served as control subjects (n = 8, control). LE, functional capillary density (FCD), and macromolecular leakage were repeatedly analyzed in the awake animals during a 24-h period using intravital fluorescence microscopy. HES 130 kD significantly reduced LPS-induced arteriolar and venular leukocyte adherence (P < 0.05), whereas NaCl resuscitation had no effect when compared with nontreated control animals. The LPS-induced decrease in FCD and increase in macromolecular leakage were also significantly attenuated by HES 130 kD but not by NaCl. Improvement of LPS-induced microcirculatory disorders by HES was unlikely the result of macro- and microhemodynamic changes because arterial blood pressure, heart rate, and venular wall shear rate did not differ between HES- and NaCl-treated animals. Thus, our study provides microhemodynamic and cellular mechanisms of HES 130 kD-mediated protection on microcirculation during endotoxemia, even when used in a clinically relevant posttreatment mode during normotensive conditions.

Met-RANTES Inhibition of Mucosal Perfusion Failure in Acute Intestinal Transplant Rejection – Role of Endothelial Cell-Leukocyte Interaction

Acute rejection-induced microvascular injury results in graft dysfunction, ultimately leading to graft loss. Infiltration of T cells and monocytes as a consequence of an enhanced endothelial cell-leukocyte interaction appears to play an important role in this deleterious process. Recruitment of these pro-inflammatory cells to the vessel wall is mediated by chemokines such as RANTES. Heterotopic small bowel transplantation was performed in rats with the fully allogeneic Brown Norway-Lewis strain combination and, as a control, the syngeneic Lewis-Lewis strain combination. Intravital microscopy was performed from postoperative day 1–7 in both groups. The percentages of perfused villi and villus stasis, mucosal and muscular functional capillary densities, red blood cell velocities, and finally, firm adherence of leukocytes in postcapillary submucosal venules were assessed. Syngeneic small bowel transplantation revealed homogeneous perfusion of villi and muscle layers over the whole study period. Allogeneic small bowel transplantation showed a decline in perfusion from postoperative day 1 until complete failure on postoperative day 7. This was accompanied by a continuous increase in endothelial cell-leukocyte interaction which reached a plateau on postoperative day 5. Met-RANTES treatment at 200 µg/day for 5 days markedly attenuated both the decrease in functional capillary density and the increased endothelial cell-leukocyte interaction in rats following allogeneic small bowel transplantation. We conclude that blocking chemokine receptors, thereby limiting endothelial cell-leukocyte interaction, may constitute a useful therapeutic approach to the prevention of microcirculatory perfusion failure in acute transplant rejection.

Adverse Effect of Heparin on Antithrombin Action during Endotoxemia: Microhemodynamic and Cellular Mechanisms

A recent clinical sepsis trial reported a significant reduction in 90-day mortality by antithrombin (AT) exclusively in the subgroup of patients without simultaneous heparin prophylaxis. Patients additionally receiving heparin did not benefit from AT treatment. Herein, we studied the microhemodynamic and cellular mechanisms of this adverse effect of heparin on AT actions by the use of intravital microscopy and granulocyte culturing. In Syrian golden hamsters normotensive endotoxemia was induced by 2 mg/kg endotoxin (LPS, E. coli) i.v. In a first group of animals, AT (AT, 250 IU/kg i.v., n = 6) was given 5 min before LPS administration. A second group of animals (Heparin + AT, n = 5) received AT (250 IU/kg i.v.) combined with unfractionated heparin (sodium heparin, 100 IU/kg/24 h, i.v.). Additional animals (LMWH + AT, n = 5) received AT (250 IU/kg i.v.) combined with LMWH (nadroparin 47.5 IU anti-Xa/kg, s.c., 2 h before LPS). LPS-treated animals, which received only saline, served as controls (control, n = 6). Using dorsal skinfold fold preparations, LPS-induced microvascular leukocyte-endothelial cell interaction (LE) and alteration of functional capillary density (FCD) were studied by intravital video fluorescence microscopy. In controls, LPS induced a massive increase in LE with a maximum at 8 h and an impressive decrease in FCD over a 24-hour period. Both LPS effects were effectively prevented by AT treatment (p < 0.01), whereas Heparin + AT and LMWH + AT animals showed microcirculatory alterations comparable to that in controls. In additional in vitro chemotaxis assays. AT blocked neutrophil chemotaxis, an effect reversed by both unfractionated heparin and LMWH. Thus, our study elucidates a relevant in vivo and in vitro unfractionated heparin and LMWH adverse effect in the microcirculatory actions of AT during endotoxemia. These results indicate that heparin should be avoided to permit AT to modulate LPS-induced inflammatory responses.

In vivo analysis of the microcirculation of osteomyocutaneous flaps using fluorescence microscopy

Previous studies have indicated that freely transferred osteomyocutaneous flaps may fail despite anastomotic patency. While microvascular dysfunction is thought to be one of the major causes for this type of flap failure, little is known of its underlying mechanisms, probably due to the lack of adequate experimental models allowing detailed intravital microcirculatory analysis. Herein we report quantitative analysis of the microcirculation of periosteum, muscle, subcutis and skin by intravital fluorescence microscopy using an osteomyocutaneous free flap model in the hindlimb of rats. The microcirculation of the different tissues was studied after microanastomotic transfer (free flap), and was compared to that after solely elevating the tissue, mimicking a pedicled osteomyocutaneous flap. Transferred flaps, which were exposed to 1 h of ischaemia during the anastomotic procedure, showed a slight but significant decrease (P< 0.05) of functional capillary density in muscle, subcutis and skin when compared with the microcirculation of pedicled flaps, while capillary diameters, red blood cell velocity and blood flow of perfused capillaries remained almost unaffected. The decrease of functional capillary density was associated by a significant (P< 0.05) inflammatory response, as indicated by the increased number of leukocytes adherent to the endothelial lining of postcapillary venules. While the functional capillary density of periosteum was not affected by the free transfer procedure, the inflammatory response was found similar when compared with that observed in muscle and subcutis. Thus, our study indicates that even after a short 1-h ischaemic time period, capillary perfusion failure and leukocyte–endothelial cell interaction are the main events, characterising microvascular dysfunction after free transfer of osteomyocutaneous flaps. Using the model described herein, intravital microscopic analysis of the microcirculation proved an appropriate tool to study the individual microvascular response after free tissue transfer, and may thus be used to evaluate the effectiveness of novel therapeutic regimens which aim at counteracting microcirculatory dysfunction in free osteomyocutaneous flaps.