We expose methods for in vivo assessment of oxygen, nitric oxide (NO), and reactive oxygen species (ROS)/reactive nitrogen species (RNS), in the microcirculation during normoxia and hypoxia. We provide an example of the related mechanisms of ROS/RNS and oxygen level in the process of regulating capillary perfusion. Namely, we discuss the real time pO2 measurements in vivo in the microvessels and tissues of the hamster cheek pouch and window chamber preparations during normoxia and hypoxia, as well as the corresponding changes in ROS/RNS in systemic blood during normoxia and hypoxia under conditions where NO availability is maximally reduced.

Polyethylene glycol (PEG) has been shown to repair cell membranes and, thus, inhibit free radical production in in vitro and in vivo models. We hypothesized that PEG and newly developed organic nitrate forms of PEG (PEG-NO) could repair endothelial dysfunction in ischemia-reperfusion (I/R) injury in the hamster cheek pouch visualized by intravital fluorescent microscopy. After treatments, we evaluated diameter and RBC velocity and flow in arterioles, as well as lipid peroxides in the systemic blood, perfused capillary length, vascular permeability, leukocyte adhesion, and amount of von Willebrand factor (vWF) in the blood after I/R injury. A control group was treated with 5,000- or 10,000-Da PEG, and three groups were treated with PG1 (1 NO molecule covalently bound to PEG, 5,170 Da), PG8 (8 NO molecules covalently bound to PEG, 11,860 Da), and PG16 (16 NO molecules covalently bound to PEG, 14,060 Da). All animals received 0.5 mg/0.5 ml. Lipid peroxides increased at 5 and 15 min of reperfusion, whereas diameter, RBC velocity, and blood flow decreased in arterioles after I/R injury. Vascular permeability, leukocyte adhesion, and vWF increased significantly. PEG and PG1 attenuated lipid peroxides and vasoconstriction during reperfusion and decreased leukocyte adhesion and vascular permeability. PG8 maintained lipid peroxides at normal levels, increased arteriolar diameter, flow, and perfused capillary length, and decreased vWF level and leukocyte adhesion (P < 0.05). PG16 was less effective than PG1 and PG8. In conclusion, PEG-NO shows promise as a compound that protects microvascular perfusion by normalizing the balance between NO level and excessive production of free radicals in endothelial cells during I/R injury.

The standardisation of manoeuvres to perform clinically discriminative microvascular flow reserve tests is still poorly developed, as well as the response analysis. The aim of this study was to establish a reproducible analysis method for the post-occlusive reactive hyperaemia (PORH) test measured using laser Doppler perfusion monitoring (LDPM). LDPM data were measured from the PORH response of 24 Fontaine class II–III peripheral atherosclerotic/arterial obstructive disease (PAOD) patients and 30 healthy subjects. The PORH response was recorded from the dorsum of the foot after 3 min of arterial occlusion at the thigh. The resulting tracings were analysed by describing their morphology through five defined parameters: resting flux (RF), time to RF level (tRF), maximum flux (MF) during reactive hyperaemia, time to maximum flux (tMF), and time to half recovery (tHR). While the time parameters were discriminative between patients and controls, flux parameters were not. The time to resting flux (tRF) led to the most discriminative model that correctly predicted 88.5% of the cases. Hence, we concluded that obtaining tRF with the presented procedures provides an optimal model to quantify the patient's microvascular condition from the PORH response.

Oxygen delivery to the tissues is crucial to survival but our understanding of the processes involved in the transport of oxygen from blood to tissue is incomplete. The aim of the present work is to illustrate a long-standing paradox regarding such transport by reporting new state-of-the-art measurements and by analyzing the results in several ways, thereby exploring possible resolutions of the paradox. Our model calculations show that slight extensions of system parameters are sufficient to overcome the apparent inconsistencies. Alternatively, so far unappreciated mild effects like flow-assisted diffusion in the interstitium will explain the supernormal diffusion of oxygen.

The role of nitric oxide ( NO) and reactive oxygen species (ROS) in regulating capillary perfusion was studied in the hamster cheek pouch model during normoxia and after 20 min of exposure to 10% O-2-90% N-2. We measured PO2 by using phosphorescence quenching microscopy and ROS production in systemic blood. Identical experiments were performed after treatment with the NO synthase inhibitor N-G-monomethyl-L- arginine (L-NMMA) and after the reinfusion of the NO donor 2,2'-(hydroxynitrosohydrazono) bis-etanamine (DETA/NO) after treatment with L-NMMA. Hypoxia caused a significant decrease in the systemic PO2. During normoxia, arteriolar intravascular PO2 decreased progressively from 47.0 +/- 3.5 mmHg in the larger arterioles to 28.0 +/- 2.5 mmHg in the terminal arterioles; conversely, intravascular PO2 was 7 - 14 mmHg and approximately uniform in all arterioles. Tissue PO2 was 85% of baseline. Hypoxia significantly dilated arterioles, reduced blood flow, and increased capillary perfusion (15%) and ROS (72%) relative to baseline. Administration of L-NMMA during hypoxia further reduced capillary perfusion to 47% of baseline and increased ROS to 34% of baseline, both changes being significant. Tissue PO2 was reduced by 33% versus the hypoxic group. Administration of DETA/NO after L-NMMA caused vasodilation, normalized ROS, and increased capillary perfusion and tissue PO2. These results indicate that during normoxia, oxygen is supplied to the tissue mostly by the arterioles, whereas in hypoxia, oxygen is supplied to tissue by capillaries by a NO concentration-dependent mechanism that controls capillary perfusion and tissue PO2, involving capillary endothelial cell responses to the decrease in lipid peroxide formation controlled by NO availability during low PO2 conditions.

The purpose of the present study was to assess whether the generalised wavelet analysis (GWA) of the leg cutaneous laser Doppler (LD) flowmotion waves recorded during baseline (Bsl) and after skin post-occlusive hyperaemia (POH) can provide information on the leg cutaneous microcirculatory adaptation to stage II peripheral arterial obstructive disease (PAOD). With this aim the flowmotion was characterised in 20 healthy subjects (HS) and 20 stage II PAOD patients by GWA of LDF tracings during Bsl and POH test. The vascular endothelial and smooth muscle function was also evaluated exploring the arm skin vasodilatory response to iontophoretically delivered acetylcholine (Ach) and sodium nitroprusside (SNP) using LD. During Bsl there was no significant difference in leg skin perfusion between HS and PAOD patients (7.3+/-5.6 vs. 5.8+/-2.9 AU, respectively). PAOD patients revealed higher peak powers in the frequency interval of 0.007-0.02 Hz (120+/-82 vs. 85+/-62 AU(2)/Hz; P < 0.05), 0.02-0.06 Hz (116+/-128 vs. 63+/-48 AU(2)/Hz, respectively; P < 0.05) and 0.06-0.2 Hz (39+/-49 vs. 14+/-10 AU(2)/Hz; P < 0.05). These flowmotion frequencies are related to vascular endothelium activity, sympathetic activity and vessel wall myogenic activity, respectively. During POH the mean peak power of the flowmotion waves increased significantly (P < 0.05) in HS respect to Bsl with the only exception of the 0.02-0.06 Hz band. In the PAOD patients, compared to Bsl the amplitude of the flowmotion waves did not significantly change during POH. In addition, the PAOD patients presented an increased time from release to peak-flux (18.25+/-15.5 vs. 2.16+/-1.28 s, respectively; P < 0.05), an increased time from release to recovery of the basal perfusion (90.26+/-39.14 vs. 26.55+/-14.05 s, respectively; P < 0.05) and a lower slope of the POH curve (10+/-15 vs. 54+/-17 degrees , respectively; P < 0.05), compared with HS. The cutaneous arm vasodilatory response to Ach and to SNP was reduced in PAOD patients in comparison with HS (P < 0.001). In conclusion, our findings showed an increased amplitude of the frequency interval 0.007-0.02, 0.02-0.06 and 0.06-0.2 Hz during Bsl in PAOD patients which did not change during the POH test. All data suggest that in stage II PAOD patients the leg skin perfusion is not impaired during Bsl because of a compensatory mechanism related to increased endothelial, myogenic and sympathetic activities. However during reactive hyperaemia these mechanisms appear to be exhausted in accordance with the reduced vasoreactivity to Ach and SNP.

Diagnostic ultrasound (US) is reported to increase intracellular oxidative stress in vitro. Increased oxidative stress mediated ischemia-reperfusion injury in the microcirculation. To examine the effects of US in hamster cheek pouch microcirculation during baseline and ischemia and reperfusion (I/R), I/R injury was provoked in the cheek pouch under "sham" (transducer off, group 1) and active US irradiation (group 2) at baseline (15 min) and at the beginning (15 min) of the reperfusion after ischemia (30 min). US transmission was delivered in the harmonic mode (2.5 MHz) with 1.3 mechanical index (MI) and 2.0 peak negative pressure. Microvascular damage was evaluated by measuring arterial diameter, red blood cell velocity, wall shear stress, permeability, perfused capillary length and adherent leukocytes in venules. Lipid peroxides were determined in the systemic blood. US increased permeability (baseline: 0.04 +/- 0.02; after US 0.30 +/- 0.04, p < 0.01) and slightly decreased capillary perfusion by 7% during baseline (p < 0.01). Arteriolar diameter (35 +/- 7 microm vs. 20 +/- 5 microm, p < 0.05), RBC velocity (2.8 +/- 0.4 mm s(-1) vs. 0.75 +/- 0.05 mm s(-1), p < 0.05) and shear stress ( 0.76 +/- 0.09 Pa vs. 0.36 +/- 0.05 Pa, p < 0.05) decreased significantly after reperfusion. These parameters increased by 40, 64 and 33%, respectively after US. Leukocyte adhesion decreased by 31 % (p < 0.05) after US and lipid peroxides decreased by 26% and 51% during baseline and 15 min of reperfusion after US, respectively. In conclusion, diagnostic US increased microvascular permeability during baseline and reperfusion. Moreover, US enhanced wall shear stress and reduced oxidative stress during postischemic reperfusion; thus, increasing capillary perfusion.

Objective: The authors investigated the effects of ACTH-(1-24) and a high-viscosity solution in the restoration of microvascular function during resuscitation. They injected N-G-monomethyl-L-arginine (L-NMMA) and superoxide dismutase ( SOD) before ACTH-(1-24) in hamsters resuscitated with the hyperviscous solution to determine the role of ROS and NO in ACTH-(1-24) protective mechanism in the cheek pouch. Hemorrhagic shock (HS) was induced by withdrawing blood to reduce mean arterial pressure (MAP) to 30 mm Hg for 45 min.Methods: Animals were injected with ACTH-(1-24) and resuscitated with dextran of low molecular weight (70 kDa) and a small amount (4%) of dextran of high molecular weight ( 500 kDa) plus ACTH( 1-24), or autologous ( shed) blood withdrawn during HS. Microvascular effects were characterized by measuring blood flow, perfused capillary length (PCL), arteriolar diameter, and red blood cell (RBC) velocity. ROS were assayed at the beginning and after 45 min of HS and after 10 and 90 min of resuscitation.Results: Resuscitation with either shed blood or dextrans 70/500 resulted in the restoration of MAP, whereas PCL, RBC velocity, and arterial diameter decreased significantly. ROS increased significantly after HS, 10 and 45 min of resuscitation. ACTH-(1-24) plus dextrans 70/500 increased MAP immediately; it increased vasodilation and PCL, and attenuated significantly ROS production and leukocyte adhesion during resuscitation. L-NMMA injected after 30 min of HS did not change the protection exerted by ACTH-(1-24) and dextrans 70/500, while SOD increased their protective effects.Conclusions: ACTH-(1-24) appears to enhance the protective effects on the endothelium exerted by increased plasma viscosity by significantly decreasing the oxidative stress and the leukocyte adhesion during resuscitation.

Aspirin that has been chemically combined with a nitric oxide (NO) donor (NCX-4016) has been shown to inhibit cyclooxygenase and prostaglandin generation while maintaining the inhibitory effects of aspirin. The possible role of reactive oxygen species (ROS) in the action of NCX-4016 in ischemia-reperfusion (I/R) has not been studied. Furthermore, we were interested in comparing the effects of a conventional NO donor [2,2'-hydroxynitrosohydrazino-bis-etanamine (DETA/NO)] and NCX-4016 at the microvascular level in the hamster cheek pouch visualized by using an intravital fluorescent microscopy technique. Microvascular injury was assessed by measuring diameter change, the perfused capillary length (PCL), and leukocyte adhesion. Animals were treated with NCX-4016 (100 mg/kg or 30 mg.kg(-1).day(-1) for 5 days po) or DETA-NO (0.5 mg/kg). Mean arterial blood pressure increased slightly but significantly after NCX-4016 treatment. During 5- and 15-min reperfusion, lipid peroxides in the systemic blood increased by 72 and 89% vs. baseline, respectively, and were still higher than in basal conditions after 30-min reperfusion in the I/R group. Pretreatment with NCX-4016 maintained ROS at normal levels; increased arteriolar diameter, blood flow, and PCL; and decreased leukocyte adhesion (P < 0.05). DETA-NO decreased ROS during 30-min reperfusion; however, later there was a significant increase during reperfusion. DETA-NO decreased leukocyte adhesion (P < 0.05) but microvascular permeability increased after 30 min of reperfusion. In conclusion, NCX-4016 attenuates oxidative stress and prevents arteriolar constriction during I/R, whereas DETA-NO increases lipid peroxides in the systemic blood and permeability after reperfusion.

Objective: The purpose of this study was to assess the efficacy of topically applied ketoprofen lysine salt (KLS), a cyclooxygenase inhibitor, against the inflammatory changes induced by interleukin-1β (IL-1β) and bradykinin (BK) in hamster cheek pouch microcirculation. In addition, we characterised the pharmacological regulation of IL-1β activity in this model.¶Materials and methods: Male Syrian hamsters were used. Microcirculation was visualised by fluorescent microscopy. Leukocyte adhesion, permeability, perfused capillary length (PCL) and capillary red blood cell (RBC) velocity were evaluated.¶Treatments: KLS (25 μg/ml/min to 1.6 mg/ml/min) was topically applied for 3 min before topically administered IL-1β (1 μg/ml) and BK (10-4 M). Monoclonal anti-mouse IL-1β receptor antagonist (200 ng/ml), BK-B2 receptor antagonist (10-6 M), PAF inhibitor (10-5 M) and cycloheximide (10 mg/ml) were added topically 15, 10, 15 and 60 min, respectively, before IL-1β (1 μg/ml).¶Results: IL-1β caused a significant increase in microvascular permeability, a decrease in capillary RBC velocity followed by increased leukocyte adhesion in postcapillary venules. BK caused a marked increase in leukocyte adhesion and no decrease in PCL and RBC velocity. Treatment with KLS significantly inhibited both the leukocyte adhesion and microvascular leakage induced by the two mediators. The inflammatory effects induced by IL-1β were reduced by blockade of IL-1β receptors and by a BK-B2 receptor antagonist but were not affected by a PAF antagonist and protein synthesis inhibition.¶Conclusions: These results demonstrate that KLS is effective in preventing early inflammatory changes induced by both IL-1β and BK in the capillary network. Prostaglandin release and BK are essential components for IL-1β mediated responses, whereas neither PAF nor new protein synthesis appear to be linked to the early inflammatory changes induced by IL-1β.

We hypothesized that during severe hemodilution (SH), i.e., hemodilution beyond 50%, the reduced conditions of shear stress result in endothelium dysfunction and subsequent vasoconstriction. To evaluate the endothelial responses associated with the reduction of hematocrit we tested the responsiveness of arterioles to acetylcholine. Infusion with high-molecular-weight dextran 500,000 following SH was characterized by laser Doppler flowmetry (LDF), total perfused capillary length (TPCL), vessel diameter, and red blood cell (RBC) velocity as visualized by fluorescence microscopy in the hamster cheek pouch. Hemodilution was performed by blood removal and simultaneous infusion of 6% dextran 70. LDF increased significantly during hemodilution in arterioles and in venules (100 +/- 20 vs 37 +/- 11 and 34.2 +/- 3.5 vs 28.6 +/- 4.0 perfusion units, PU, respectively). During the final step of hemodilution LDF decreased significantly to 12 +/- 4.5 PU in arterioles and 6.2 +/- 1.5 PU in venules, which correlated with the decrease of arteriolar RBC velocity. Arterioles constricted and TPCL decreased significantly (-35 +/- 5.3%). Hyperviscosity infusion significantly raised arteriolar and venular LDF to 184 +/- 15 and 40.2 +/- 3.5 PU, arterioles dilated, RBC velocity, TPCL, and mean blood pressure. There was an impairment of endothelial-dependent dilation that was not present in the group with dextran 500, which suggests that viscosity was involved in the development of vasoconstriction during SH. In conclusion, an increase in plasma viscosity has beneficial effects on the microcirculation during SH that might preserve arteriolar endothelium and capillary perfusion.

Systemic hypoxia (8%, 11% and 15% oxygen gas mixture inspiration) has been shown to increase the frequency of arteriolar rhythmic diameter changes in hamster skeletal muscle microcirculation. The effects of phentolamine on vasomotion frequency during systemic hypoxia were studied in Syrian hamsters implanted with a plastic chamber in the dorsum skin. Phentolamine(50 mug/100 g body wt.) was injected intravenously before the 20-min exposure to 11% oxygen gas mixture. The microvessels were studied with a fluorescent microscopy technique, using fluorescein isothiocyanate bound to dextran (mol. wt. 150,000) as a tracer. Vessel diameters were measured with a: shearing method. Fourier transform and autoregressive modeling were used to assess the time variant features of diameter changes.Under baseline conditions, the arterioles were characterized by rhythmic diameter changes with fundamental frequency related to vessel size. The terminal branchings were dominated by order 3 vessel activity (frequency: 0.08-0.16 Hz) spreading downstream to all daughter arterioles. Systemic hypoxia caused an increase in vasomotion frequency of order 3 arterioles up to 0.3-0.5 Hz (average: 0.40 +/- 0.06 Hz) and a significant decrease in mean diameter(-28 +/- 5%). Phentolamine completely suppressed the rhythmic changes in diameter of order 3 arterioles that dilated significantly (+30 +/- 4%).Therefore, the effects of systemic hypoxia on arteriolar vasomotion appear to be triggered by an increase in sympathetic nervous discharge that induces a rise in frequency up to 0.3-0.5 Hz. (C) 2001 Elsevier Science B.V. All rights reserved.

Aims/hypothesis. Treatment with intravenous glucose-insulin-potassium has beneficial effects in reperfused patients, reducing mortality in patients with myocardial infarction by 28%. We hypothesized that insulin response to glucose-insulin-potassium infusion might lead to vasodilation in ischemia/reperfusion (I/R). Hyperglycaemia and hyperinsulinaemia determine oxidative stress. We therefore investigated the microcirculatory changes following I/R after glucose-insulin-potassium or in association with glucose-insulin-potassium dipyridamole in hamster cheek pouch.Methods. The control (I/R), glucose-insulin-potassium groups with and without dipyridamole were treated with saline, 300 g/l, 50 U/l insulin and 80 meq/l KCl infused at 0.2 ml (.) 100 g(-1) (.) h(-1), and GIK plus dipyridamole (0.084 mg (.) 100 g(-1) intravenously) at beginning, 30 min before ischaemia, and continuing through reperfusion. We measured microvessel diameter changes, arteriolar red blood cell velocity, permeability increase, capillary perfused length, leukocyte and platelet adhesion.Results. Hyperglycaemia and hyperinsulinaemia did not cause vasodilation whereas in the glucose-insulin-potassium group with dipyridamole there was a marked arterial vasodilation with increased red blood cell velocity and perfused capillary length at reperfusion. Glucose-insulin-potassium infusion reversed the arterial vasoconstriction caused by I/R at reperfusion. Adhering leukocytes to venules decreased by 56 and 86% while platelets adhering to microvessels was reduced by 52 and 72% at reperfusion in glucose-insulin-potassium groups with and without dipyridamole, respectively. The permeability was decreased by GIK and completely suppressed by GIKD after I/R.Conclusion hypothesis. We demonstrated that GIK, when used in combination with dipyridamole, had beneficial effects on the capillary perfusion against I/R-induced injury. There was a marked reduction of leukocyte and platelet adhesion that can be explained by the antioxidant properties of dipyridamole.

S: 12th International Contact Dermatitis Symposium and 1st International Symposium on the Prevention of Occupational Skin Disease in Hairdressers San Francisco, CA, USA October 14-18, 1999: Part 3 (Continued From December 2000 Issue): PDF Only

Ischemia shifts the anticoaugulant/procoagulant balance of the endothelium in favor of activation of coagulation. We studied whether cheek pouch microcirculation of leukopenic hamsters was protected by tissue plasminogen activator (tPA) (50 microg/100 g body wt) against ischemia-reperfusion injury. Adherent leukocytes, total perfused capillary length (PCL), permeability increase, and arteriolar and venular red blood cell (RBC) velocity were investigated by fluorescence microscopy. Measurements were made at control, 30 or 60 min of ischemia, and at 30 or 60 min of reperfusion. Hamsters were made leukopenic by treatment with cyclophosphamide (20 mg/100 g body wt ip, 4 days before the experiment), which decreased circulating leukocyte count by 85-90%. Leukopenic hamsters undergoing 30 min of ischemia followed by 30 min of reperfusion showed no significant decrease in PCL or increased permeability. Leukopenic hamsters undergoing 60 min of ischemia followed by 60 min of reperfusion presented a significant decrease in microvascular perfusion where PCL was 28 +/- 7% of baseline, low-flow conditions, and increased permeability. In leukopenic hamsters treated with tPA there was complete protection of capillary perfusion with no significant changes in permeability or arteriolar and venular RBC velocity. In conclusion, thrombus formation may be an additional and independent factor that with leukocyte-mediated mechanisms determines ischemia-reperfusion injury.

Flowmotion was characterized in healthy controls and 61 Raynaud's phenomenon (RP) patients by spectral analysis of laser-Doppler perfusion monitoring (LDPM) tracings. Healthy subjects flowmotion patterns revealed a main frequency of 3 cycles per min (cpm) with another low frequency and heart rate synchronous components. A first group of RP patients presented a low frequency and heart rate frequency component but no significant difference in blood flow. The second group presented the predominating heart rate related frequency with low microvascular perfusion. The third group presented a flowmotion pattern with overlapping of heart rate and low frequency components. Patients with primary and secondary RP show specific changes in flowmotion, probably related to increased sympathetic nervous activity or vessel wall alterations causing disappearance of arteriolar tone and impairment of microvascular perfusion. The group of patients with overlapping frequency components presents an intermediate flowmotion pattern indicating a different grade of alterations in microvasculature.

An enhanced high-resolution laser Doppler imager (EHR-LDI), configured to fit the demands of a measurement area containing separate microvessels, was evaluated for perfusion measurements in hamster cheek pouch preparations during ischemia, reperfusion, and pharmacologically induced vasodilation and vasoconstriction. Measurements in separate microvessels where the laser beam was smaller than the vessel diameter were referred to as red blood cell (RBC) velocity estimates, as previously validated in vitro, whereas a relative flow index, RFI (mean RBC velocity/tissue area), was introduced as a volumetric flow measure. Microvessel diameter and RBC velocity changes during ischemia, reperfusion, as well as during vasoconstriction and vasodilation correlated to the data obtained from the microscope. Correspondingly, during the described provocations anticipated volumetric flow changes were registered as changes in the RFI. When data on intravessel RBC velocity profiles are presented they reflect a parabolic flow profile usually seen in this size microvessel. The EHR-LDI appears a promising tool for investigation of the microvasculature, as it almost simultaneously provides information on relative changes of both in vivo RBC velocity and volumetric flow (RFI), although the latter estimate needs to be further refined.