Identifying a sublingual triangle as the ideal site for assessment of sublingual microcirculation.

The sublingual mucosa is a commonly used intraoral location for identifying microcirculatory alterations using handheld vital microscopes (HVMs). The anatomic description of the sublingual cave and its related training have not been adequately introduced. The aim of this study was to introduce anatomy guided sublingual microcirculatory assessment. Measurements were acquired from the floor of the mouth using incident dark-field (IDF) imaging before (T0) and after (T1) sublingual cave anatomy instructed training. Instructions consists of examining a specific region of interested identified through observable anatomical structures adjacent and bilaterally to the lingual frenulum which is next to the sublingual papilla. The anatomical location called the sublingual triangle, was identified as stationed between the lingual frenulum, the sublingual fold and ventrally to the tongue. Small, large, and total vessel density datasets (SVD, LVD and TVD respectively) obtained by non-instructed and instructed measurements (NIN (T0) and IM (T1) respectively) were compared. Microvascular structures were analyzed, and the presence of salivary duct-related microcirculation was identified. A total of 72 video clips were used for analysis in which TVD, but not LVD and SVD, was higher in IM compared to NIM (NIM vs. IM, 25 ± 2 vs. 27 ± 3 mm/mm2 (p = 0.044), LVD NIM vs. IM: 7 ± 1 vs. 8 ± 1mm/mm2 (p = 0.092), SVD NIM vs. IM: 18 ± 2 vs. 20 ± 3 mm/mm2 (p = 0.103)). IM resulted in microcirculatory assessments which included morphological properties such as capillaries, venules and arterioles, without salivary duct-associated microcirculation. The sublingual triangle identified in this study showed consistent network-based microcirculation, without interference from microcirculation associated with specialized anatomic structures. These findings suggest that the sublingual triangle, an anatomy guided location, yielded sublingual based measurements that conforms with international guidelines. IM showed higher TVD values, and future studies are needed with larger sample sizes to prove differences in microcirculatory parameters.

Morphologic Mapping of the Sublingual Microcirculation in Healthy Volunteers.

PURPOSE: Monitoring the sublingual and oral microcirculation (SM-OM) using hand-held vital microscopes (HVMs) has provided valuable insight into the (patho)physiology of diseases. However, the microvascular anatomy in a healthy population has not been adequately described yet. METHODS: Incident dark field-based HVM imaging was used to visualize the SM-OM. First, the SM was divided into four different fields; Field-a (between incisors-lingua), Field-b (between the canine-first premolar-lingua), Field-c (between the first-second premolar-lingua), Field-d (between the second molar-wisdom teeth-lingua). Second, we investigated the buccal area, lower and upper lip. Total/functional vessel density (TVD/FCD), focus depth (FD), small vessel mean diameters (SVMDs), and capillary tortuosity score (CTS) were compared between the areas. RESULTS: Fifteen volunteers with a mean age of 29 ± 6 years were enrolled. No statistical difference was found between the sublingual fields in terms of TVD (p = 0.30), FCD (p = 0.38), and FD (p = 0.09). SVMD was similar in Field-a, Field-b, and Field-c (p = 0.20-0.30), and larger in Field-d (p < 0.01, p = 0.015). The CTS of the buccal area was higher than in the lips. CONCLUSION: The sublingual area has a homogenous distribution in TVD, FCD, FD, and SVMD. This study can be a description of the normal microvascular anatomy for future researches regarding microcirculatory assessment.

Microcirculatory alterations in critically ill COVID-19 patients analyzed using artificial intelligence.

BACKGROUND: The sublingual microcirculation presumably exhibits disease-specific changes in function and morphology. Algorithm-based quantification of functional microcirculatory hemodynamic variables in handheld vital microscopy (HVM) has recently allowed identification of hemodynamic alterations in the microcirculation associated with COVID-19. In the present study we hypothesized that supervised deep machine learning could be used to identify previously unknown microcirculatory alterations, and combination with algorithmically quantified functional variables increases the model's performance to differentiate critically ill COVID-19 patients from healthy volunteers. METHODS: Four international, multi-central cohorts of critically ill COVID-19 patients and healthy volunteers (n = 59/n = 40) were used for neuronal network training and internal validation, alongside quantification of functional microcirculatory hemodynamic variables. Independent verification of the models was performed in a second cohort (n = 25/n = 33). RESULTS: Six thousand ninety-two image sequences in 157 individuals were included. Bootstrapped internal validation yielded AUROC(CI) for detection of COVID-19 status of 0.75 (0.69-0.79), 0.74 (0.69-0.79) and 0.84 (0.80-0.89) for the algorithm-based, deep learning-based and combined models. Individual model performance in external validation was 0.73 (0.71-0.76) and 0.61 (0.58-0.63). Combined neuronal network and algorithm-based identification yielded the highest externally validated AUROC of 0.75 (0.73-0.78) (P < 0.0001 versus internal validation and individual models). CONCLUSIONS: We successfully trained a deep learning-based model to differentiate critically ill COVID-19 patients from heathy volunteers in sublingual HVM image sequences. Internally validated, deep learning was superior to the algorithmic approach. However, combining the deep learning method with an algorithm-based approach to quantify the functional state of the microcirculation markedly increased the sensitivity and specificity as compared to either approach alone, and enabled successful external validation of the identification of the presence of microcirculatory alterations associated with COVID-19 status.

Increased Hepatic Microvascular Density, Oxygenation, and VEGF in the Hypertrophic Lobe following Portal Vein Embolization in Rabbits.

INTRODUCTION: The microvascular events following portal vein embolization (PVE) are poorly understood despite the pivotal role of the microcirculation in liver regeneration and tumor progression. We aimed to assess the changes in hepatic microvascular perfusion and neo-angiogenesis after experimental PVE. METHODS: PVE of the cranial liver lobes was performed in 12 New Zealand White rabbits divided into 2 groups of permanent (P-PVE) and reversible PVE (R-PVE), respectively. Hepatobiliary scintigraphy and CT were used to evaluate hepatic function and volume. Hepatic microcirculation was assessed using a handheld vital microscope (Cytocam) to measure microvascular density (total vessel density; TVD) before PVE, right after PVE, and 20 min after PVE, as well as at 14 days (D14 post-PVE) and 35 days (D35 post-PVE). Additionally, on D35, microvascular PO2 and liver parenchymal VEGF were assessed. RESULTS: Eleven rabbits were included after PVE (R-PVE, n = 5; P-PVE, n = 6). TVD in the nonembo-lized (hypertrophic) lobes was higher than in the embolized (atrophic) lobes of the P-PVE group at D35 post-PVE (36.7 ± 7.2 vs. 23.4 ± 4.9 mm/mm2; p < 0.05). In the R-PVE group, TVD in the nonembolized lobes was not increased at D35. Function and volume were increased in the nonembolized lobes of the P-PVE group compared to the embolized lobes, but not in the R-PVE group. Likewise, the mmicrovascular PO2 and VEGF staining rate were higher in the nonembolized lobes of the P-PVE group at D35 post-PVE. DISCUSSION/CONCLUSION: Successful volumetric and functional hypertrophy of the nonembolized lobe was accompanied by microvascular alterations featuring increased neo-angiogenesis, microvascular density, and microvascular oxygen pressure following P-PVE.

Capillary Leukocytes, Microaggregates, and the Response to Hypoxemia in the Microcirculation of Coronavirus Disease 2019 Patients.

OBJECTIVES: In this study, we hypothesized that coronavirus disease 2019 patients exhibit sublingual microcirculatory alterations caused by inflammation, coagulopathy, and hypoxemia. DESIGN: Multicenter case-controlled study. SETTING: Two ICUs in The Netherlands and one in Switzerland. PATIENTS: Thirty-four critically ill coronavirus disease 2019 patients were compared with 33 healthy volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The microcirculatory parameters quantified included total vessel density (mm × mm-2), functional capillary density (mm × mm-2), proportion of perfused vessels (%), capillary hematocrit (%), the ratio of capillary hematocrit to systemic hematocrit, and capillary RBC velocity (µm × s-1). The number of leukocytes in capillary-postcapillary venule units per 4-second image sequence (4 s-1) and capillary RBC microaggregates (4 s-1) was measured. In comparison with healthy volunteers, the microcirculation of coronavirus disease 2019 patients showed increases in total vessel density (22.8 ± sd 5.1 vs 19.9 ± 3.3; p < 0.0001) and functional capillary density (22.2 ± 4.8 vs 18.8 ± 3.1; p < 0.002), proportion of perfused vessel (97.6 ± 2.1 vs 94.6 ± 6.5; p < 0.01), RBC velocity (362 ± 48 vs 306 ± 53; p < 0.0001), capillary hematocrit (5.3 ± 1.3 vs 4.7 ± 0.8; p < 0.01), and capillary-hematocrit-to-systemic-hematocrit ratio (0.18 ± 0.0 vs 0.11 ± 0.0; p < 0.0001). These effects were present in coronavirus disease 2019 patients with Sequential Organ Failure Assessment scores less than 10 but not in patients with Sequential Organ Failure Assessment scores greater than or equal to 10. The numbers of leukocytes (17.6 ± 6.7 vs 5.2 ± 2.3; p < 0.0001) and RBC microaggregates (0.90 ± 1.12 vs 0.06 ± 0.24; p < 0.0001) was higher in the microcirculation of the coronavirus disease 2019 patients. Receiver-operating-characteristics analysis of the microcirculatory parameters identified the number of microcirculatory leukocytes and the capillary-hematocrit-to-systemic-hematocrit ratio as the most sensitive parameters distinguishing coronavirus disease 2019 patients from healthy volunteers. CONCLUSIONS: The response of the microcirculation to coronavirus disease 2019-induced hypoxemia seems to be to increase its oxygen-extraction capacity by increasing RBC availability. Inflammation and hypercoagulation are apparent in the microcirculation by increased numbers of leukocytes and RBC microaggregates.

Goal-directed fluid therapy vs. low central venous pressure during major open liver resections (GALILEO): a surgeon- and patient-blinded randomized controlled trial.

BACKGROUND: Low central venous pressure (low-CVP) is the clinical standard for fluid therapy during major liver surgery. Although goal-directed fluid therapy (GDFT) has been associated with reduced morbidity and mortality in major abdominal surgery, concerns remain on blood loss when applying GDFT in liver surgery. This randomized trial compared outcomes of low-CVP and GDFT during major liver resections. METHODS: In this surgeon- and patient-blinded RCT, patients undergoing major open liver resections (≥3 segments) were randomized between low-CVP (n = 20) or GDFT (n = 20). Primary outcome was intraoperative blood loss. Secondary outcomes included the quality of the surgical field (VAS scale 0 (worst)-100 (best)) and major morbidity (≥grade 3 Clavien-Dindo). RESULTS: During surgery, CVP was 3 ± 2 mmHg in the low-CVP group vs. 7 ± 3 mmHg in the GDFT group (P < 0.001). Blood loss (1425 vs. 1275 mL; P = 0.640) and the rate of major morbidity (40% vs. 50%, P = 0.751), did not differ between low-CVP and GDFT, respectively. The quality of the surgical field was comparable between groups (low-CVP 83% vs. GDFT 80%, P = 0.955). CONCLUSION: In major open liver resections, GDFT was not associated with differences in intraoperative blood loss, major morbidity or quality of the surgical field, compared to low-CVP. Larger RCTs are needed to confirm this finding. Registration number: NTR5821 (www.trialregister.nl).

Automated Algorithm Analysis of Sublingual Microcirculation in an International Multicentral Database Identifies Alterations Associated With Disease and Mechanism of Resuscitation.

OBJECTIVES: Reliable automated handheld vital microscopy image sequence analysis and the identification of disease states and effects of therapy are prerequisites for the routine use of quantitative sublingual microcirculation measurements at the point-of-care. The present study aimed to clinically validate the recently introduced MicroTools software in a large multicentral database of perioperative and critically ill patients and to use this automatic algorithm to data-mine and identify the sublingual microcirculatory variable changes in response to disease and therapy. DESIGN: Retrospective algorithm-based image analysis and data-mining within a large international database of sublingual capillary microscopy. Algorithm-based analysis was compared with manual analysis for validation. Thereafter, MicroTools was used to identify the functional microcirculatory alterations associated with disease conditions and identify therapeutic options for recruiting functional microcirculatory variables. SETTING: Ten perioperative/ICU/volunteer studies in six international teaching hospitals. PATIENTS: The database encompass 267 adult and pediatric patients undergoing surgery, treatment for sepsis, and heart failure in the ICU and healthy volunteers. INTERVENTIONS: Perioperative and ICU standard of care. MEASUREMENTS AND MAIN RESULTS: One thousand five hundred twenty-five handheld vital microscopy image sequences containing 149,257 microscopy images were analyzed. 3.89 × 10 RBC positions were tracked by the algorithm in real time, and offline manual analysis was performed. Good correlation and trending ability were found between manual and automatic total and functional capillary density (r = 0.6-0.8; p < 0.0001). RBC tracking within the database demonstrated changes in functional capillary density and/or RBC velocity in septic shock, heart failure, hypovolemia, obstructive shock, and hemodilution and thus detected the presence of a disease condition. Therapies recruiting the microcirculatory diffusion and convection capacity associated with systemic vasodilation and an increase in cardiac output were separately identified. CONCLUSIONS: Algorithm-based analysis of the sublingual microcirculation closely matched manual analysis across a broad spectrum of populations. It successfully identified a methodology to quantify microcirculatory alterations associated with disease and the success of capillary recruitment, improving point-of-care application of microcirculatory-targeted resuscitation procedures.

Leukocyte-Endothelium Interaction in the Sublingual Microcirculation of Coronary Artery Bypass Grafting Patients.

OBJECTIVE: The aim of this study was to apply an innovative methodology to incident dark-field (IDF) imaging in coronary artery bypass grafting (CABG) patients for the identification and quantification of rolling leukocytes along the sublingual microcirculatory endothelium. METHODS: This study was a post hoc analysis of a prospective study that evaluated the perioperative course of the sublingual microcirculation in CABG patients. Video images were captured using IDF imaging following the induction of anesthesia (T0) and cardiopulmonary bypass (CPB) (T1) in 10 patients. Rolling leukocytes were identified and quantified using frame averaging, which is a technique that was developed for correctly identifying leukocytes. RESULTS: The number of rolling leukocytes increased significantly from T0 (7.5 [6.4-9.1] leukocytes/capillary-postcapillary venule/4 s) to T1 (14.8 [13.2-15.5] leukocytes/capillary-postcapillary venule/4 s) (p < 0.0001). A significant increase in systemic leukocyte count was also detected from 7.4 ± 0.9 × 109/L (preoperative) to 12.4 ± 4.4 × 109/L (postoperative) (p < 0.01). CONCLUSION: The ability to directly visualize leukocyte-endothelium interaction using IDF imaging facilitates the diagnosis of a systemic inflammatory response after CPB via the identification of rolling leukocytes. Integration of the frame averaging algorithm into the software of handheld vital microscopes may enable the use of microcirculatory leukocyte count as a real-time parameter at the bedside.

Intraoperative Imaging Techniques to Visualize Hepatic (Micro)Perfusion: An Overview.

The microcirculation plays a crucial role in the distribution of perfusion to organs. Studies have shown that microcirculatory dysfunction is an independent predictor of morbidity and mortality. Hence, assessment of liver perfusion offers valuable information on the (patho)physiological state of the liver. The current review explores techniques in perfusion imaging that can be used intraoperatively. Available modalities include dynamic contrast-enhanced ultrasound, handheld vital microscopes, indocyanine green fluorescence angiography, and laser contrast speckle imaging. Dynamic contrast-enhanced ultrasound relays information on deep tissue perfusion and is a commonly used technique to assess tumor perfusion. Handheld vital microscopes provide direct visualization of the sinusoidal architectural structure of the liver, which is a unique feature of this technique. Intraoperative fluorescence imaging uses indocyanine green, a dye that is administered intravenously to visualize microvascular perfusion when excited using near-infrared light. Laser speckle contrast imaging produces non-contact large surface-based tissue perfusion imaging free from movement- or pressure-related artefacts. In this review, we discuss the intrinsic advantages and disadvantages of these techniques and their clinical and/or scientific applications.

Glycocalyx Degradation Is Independent of Vascular Barrier Permeability Increase in Nontraumatic Hemorrhagic Shock in Rats.

BACKGROUND: Glycocalyx shedding after traumatic hemorrhagic or septic shock, as well as different resuscitation fluids, has been causally linked to increased vascular barrier permeability (VBP) resulting in tissue edema. In nontraumatic hemorrhagic shock (NTHS), it remains questionable whether glycocalyx degradation in itself results in an alteration of VBP. The composition of fluids can also have a modulatory effect on glycocalyx shedding and VBP. We hypothesized that the shedding of the glycocalyx during NTHS has little effect on VBP and that the composition of fluids can modulate these effects. METHODS: Fully instrumented Wistar-albino rats were subjected to a pressure-controlled NTHS (mean arterial pressure of 30 mm Hg) for 60 minutes. Animals were fluid resuscitated with Ringer's acetate, balanced hydroxyethyl starch (HES) solution, or 0.9% normal saline to a mean arterial pressure of 80 mm Hg and compared with shams or nonresuscitated NTHS. Glycocalyx shed products were determined at baseline and 60 minutes after fluid resuscitation. Skeletal muscle microcirculation was visualized using handheld vital microscopy. VBP changes were assessed using plasma decay of 3 fluorescent dyes (40- and 500-kDa dextran and 70-kDa albumin), Evans blue dye exclusion, intravital fluorescence microscopy, and determination of tissue edema (wet/dry weight ratio). RESULTS: All glycocalyx shedding products were upgraded as a result of NTHS. Syndecan-1 significantly increased in NTHS (mean difference, -1668; 95% confidence interval [CI], -2336 to -1001; P < .0001), balanced crystalloid (mean difference, -964.2; 95% CI, -1492 to -436.4; P = .0001), and HES (mean difference, -1030; 95% CI, -1594 to -465.8; P = .0001) groups at the end of the experiment compared to baseline. Hyaluronan levels were higher at the end of the experiment in nonresuscitated NTHS (-923.1; 95% CI, -1216 to -630; P = .0001) and balanced crystalloid (-1039; 95% CI, -1332 to -745.5; P = .0001) or HES (-394.2; 95% CI, -670.1 to -118.3; P = .0027) groups compared to controls. Glycocalyx shedding resulted in microcirculation alterations as observed by handheld video microscopy. Total vessel density was altered in the normal saline (mean difference, 4.092; 95% CI, 0.6195-7.564; P = .016) and hemorrhagic shock (mean difference, 5.022; 95% CI, 1.55-8.495; P = .0024) groups compared to the control group, as well as the perfused vessel density and mean flow index. Despite degradation of endothelial glycocalyx, VBP as determined by 4 independent assays remained intact and continued to be so following fluid resuscitation. CONCLUSIONS: NTHS induced glycocalyx shedding and microcirculation alterations, without altering VBP. Fluid resuscitation partially restored the microcirculation without altering VBP. These results challenge the concept that the glycocalyx barrier is a significant contributor to VBP.

Assessment of hepatic microvascular flow and density in patients undergoing preoperative portal vein embolization.

BACKGROUND: The microvascular effects occurring after unilateral preoperative portal vein embolization (PVE) are poorly understood. The aim of this study was to assess the microvascular changes in the embolized and the non-embolized lobes after right PVE. METHODS: Videos of the hepatic microcirculation in patients undergoing right hemihepatectomy following PVE were recorded using a handheld vital microscope (Cytocam) based on incident dark field imaging. Hepatic microcirculation was measured in the embolized and the non-embolized lobes at laparotomy, 3-6 weeks after PVE. The following microcirculatory parameters were assessed: total vessel density (TVD), microcirculatory flow index (MFI), proportion of perfused vessel (PPV), perfused vessel density (PVD), sinusoidal diameter (SinD) and the absolute red blood cell velocity (RBCv). RESULTS: 16 patients after major liver resection were included, 8 with and 8 without preoperative PVE. Microvascular density parameters were higher in the non-embolized lobes when compared to the embolized lobes (TVD: 40.3 ± 8.9 vs. 26.8 ± 4.6 mm/mm2 (p < 0.003), PVD: 40.3 ± 8.8 vs. 26.7 ± 4.7 mm/mm2 (p < 0.002), SinD: 9.2 ± 1.7 vs. 6.3 ± 0.8 µm (p < 0.040)). RBCv, PPV and the MFI were not significantly different. CONCLUSION: The non-embolized lobe has a significantly higher microvascular density, however without differences in microvascular flow. These findings indicate increased angiogenesis in the hypertrophic lobe.

Intraoperative Incident Dark Field Imaging of the Human Peritoneal Microcirculation.

BACKGROUND/AIMS: This study describes the peritoneal microcirculation, compares quantitative parameters and angioarchitecture to the standard of sublingual microcirculatory assessment, and determines the practical feasibility of this method. METHODS: Incident dark field imaging was performed of the peritoneum and sublingually to determine angioarchitecture, total and perfused vessel density (TVD and PVD), the proportion of perfused vessels (PPV), the microvascular flow index (MFI) and image acquisition time. RESULTS: Peritoneal angioarchitecture was characterized by a quadrangular network of longitudinally oriented capillaries, often flanked by fat cells. Differences between peritoneal and sublingual microcirculation were observed with regard to TVD (peritoneum 12 mm/mm2 [95% CI 10-14] vs. sublingual 23 mm/mm2 [95% CI 21-25]; p < 0.0001), PVD (peritoneum 11 mm/mm2 [95% CI 9-13] vs. sublingual 23 mm/mm2 [95% CI 21-25]; p < 0.0001), PPV (peritoneum 88% [95% CI 79-97] vs. sublingual 99% [95% CI 99-100]; p = 0.014), and MFI (peritoneum 3 [IQR 2.3-3.0] vs. sublingual 3 [IQR 3.0-3.0]; p = 0.012). There was no difference in image acquisition time (peritoneum 2: 34 min [95% CI 1: 49-3: 19] vs. sublingual 2: 38 [95% CI 1: 37-3: 32]; p = 0.916). CONCLUSION: The peritoneal microcirculation was characterized by a low capillary density and a distinctive angioarchitecture. The possibility of peri-toneal microcirculatory assessment offers promise for the study of peritoneal (patho-)physiology and (monitoring or detection of) associated diseases.

Circulating microaggregates during cardiac surgery precedes postoperative stroke.

Postoperative stroke and encephalopathy are potentially serious complications associated with coronary artery bypass grafting. In this case report a 78-year-old male patient receiving routine elective cardiac surgery presented with microaggregations in the sublingual microcirculation while on cardiopulmonary bypass that was undetected by routine intraoperative anticoagulation assessment. Microaggregates identified using video microscopy on his sublingual microcirculation during the procedure preceded a stroke postoperatively. Postoperative cerebral and carotid artery examination with computed tomography scanning revealed a left watershed cerebral infarct with carotid stenosis. This report presents intraoperative microcirculation-based evidence suggesting that observations of microaggregations, otherwise undetected by conventional anticoagulation assessment techniques, could serve as an early warning in elderly patients at high risk for postoperative cerebrovascular events.

The microcirculation in the first days of ICU admission in critically ill COVID-19 patients is influenced by severity of disease.

The objective of this study was to investigate the relationship between sublingual microcirculatory parameters and the severity of the disease in critically ill coronavirus disease 2019 (COVID-19) patients in the initial period of Intensive Care Unit (ICU) admission in a phase of the COVID-19 pandemic where patients were being treated with anti-inflammatory medication. In total, 35 critically ill COVID-19 patients were included. Twenty-one critically ill COVID-19 patients with a Sequential Organ Failure Assessment (SOFA) score below or equal to 7 were compared to 14 critically ill COVID-19 patients with a SOFA score exceeding 7. All patients received dexamethasone and tocilizumab at ICU admission. Microcirculatory measurements were performed within the first five days of ICU admission, preferably as soon as possible after admission. An increase in diffusive capacity of the microcirculation (total vessel density, functional capillary density, capillary hematocrit) and increased perfusion of the tissues by red blood cells was found in the critically ill COVID-19 patients with a SOFA score of 7-9 compared to the critically ill COVID-19 patients with a SOFA score ≤ 7. No such effects were found in the convective component of the microcirculation. These effects occurred in the presence of administration of anti-inflammatory medication.

Real-time observation of microcirculatory leukocytes in patients undergoing major liver resection.

Ischemia/reperfusion injury and inflammation are associated with microcirculatory dysfunction, endothelial injury and glycocalyx degradation. This study aimed to assess microcirculation in the sublingual, intestinal and the (remnant) liver in patients undergoing major liver resection, to define microcirculatory leukocyte activation and its association with glycocalyx degradation. In this prospective observational study, the microcirculation was assessed at the beginning of surgery (T0), end of surgery (T1) and 24 h after surgery (T2) using Incident Dark Field imaging. Changes in vessel density, blood flow and leukocyte behaviour were monitored, as well as clinical parameters. Syndecan-1 levels as a parameter of glycocalyx degradation were analysed. 19 patients were included. Sublingual microcirculation showed a significant increase in the number of rolling leukocytes between T0 and T1 (1.5 [0.7-1.8] vs. 3.7 [1.7-5.4] Ls/C-PCV/4 s respectively, p = 0.001), and remained high at T2 when compared to T0 (3.8 [3-8.5] Ls/C-PCV/4 s, p = 0.006). The microvascular flow decreased at T2 (2.4 ± 0.3 vs. baseline 2.8 ± 0.2, respectively, p < 0.01). Duration of vascular inflow occlusion was associated with significantly higher numbers of sublingual microcirculatory rolling leukocytes. Syndecan-1 increased from T0 to T1 (42 [25-56] vs. 107 [86-164] ng/mL, p < 0.001). The microcirculatory perfusion was characterized by low convection capacity and high number of rolling leukocytes. The ability to sublingually monitor the rolling behaviour of the microcirculatory leukocytes allows for early identification of patients at risk of increased inflammatory response following major liver resection.

Hemodilution causes glycocalyx shedding without affecting vascular endothelial barrier permeability in rats.

BACKGROUND: The consequences of acute normovolemic hemodilution (ANH) following different types of fluids on the different components of the glycocalyx and on vascular barrier permeability (VBP) remain unknown. AIM: The aim of the study was to investigate whether the microcirculatory disruption and glycocalyx shedding induced by ANH alters VBP and whether this is affected by the composition and volume of the resuscitation fluid. MATERIALS AND METHODS: Anesthetized Wistar albino rats (n=24) underwent stepwise ANH at hematocrit levels of 35%, 25%, 20%, and 15% induced by the exchange of blood with 6% balanced hydroxyethyl starch (1:1), balanced crystalloid (1:3), and normal saline (NS) (1:3). Glycocalyx-shed products were measured at each level of hemodilution. VBP was reflected in the decay of fluorescence dyes of different molecular size and their plasma retention ratios. Edema was assessed by measuring organ water content and muscle microcirculation by hand-held videomicroscopy. RESULTS: NS caused increased degradation of heparan sulfate and hyaluronan compared with the control group (P=0.003, P=0.004, respectively). Neither VBP nor tissue edema was affected by the fluid used. The total and perfused vessel densities within the microcirculation of muscle tissue decreased at hematocrit 15% in the balanced crystalloid (P=0.02) and NS groups only (P<0.0001, P=0.0003, respectively) compared with baseline. CONCLUSIONS: Balanced colloid solution preserved the glycocalyx layer better than balanced and unbalanced crystalloid solutions while maintaining the microcirculatory function associated with an improved total intravascular volume. Among the fluids tested, NS caused the most microcirculatory alterations. While ANH caused the degradation of glycocalyx components regardless of fluid, it did not disrupt the vascular barrier as indicated by macromolecular leakage. RELEVANCE FOR PATIENTS: The results of this study provide insight into the choice of fluid for optimal perioperative fluid management and the consequences of fluid type on the vascular barrier, glycocalyx, and microcirculation.

Interpatient heterogeneity in hepatic microvascular blood flow during vascular inflow occlusion (Pringle manoeuvre).

BACKGROUND: Vascular inflow occlusion (VIO) during liver resections (Pringle manoeuvre) can be applied to reduce blood loss, however may at the same time, give rise to ischemia-reperfusion injury (IRI). The aim of this study was to assess the characteristics of hepatic microvascular perfusion during VIO in patients undergoing major liver resection. METHODS: Assessment of hepatic microcirculation was performed using a handheld vital microscope (HVM) at the beginning of surgery, end of VIO (20 minutes) and during reperfusion after the termination of VIO. The microcirculatory parameters assessed were: functional capillary density (FCD), microvascular flow index (MFI) and sinusoidal diameter (SinD). RESULTS: A total of 15 patients underwent VIO; 8 patients showed hepatic microvascular perfusion despite VIO (partial responders) and 7 patients showed complete cessation of hepatic microvascular perfusion (full responders). Functional microvascular parameters and blood flow levels were significantly higher in the partial responders when compared to the full responders during VIO (FCD: 0.84±0.88 vs. 0.00±0.00 mm/mm2, P<0.03, respectively, and MFI: 0.69-0.22 vs. 0.00±0.00, P<0.01, respectively). CONCLUSIONS: An interpatient heterogeneous response in hepatic microvascular blood flow was observed upon VIO. This may explain why clinical strategies to protect the liver against IRI lacked consistency.

Poor perfusion of the microvasculature in peritoneal metastases of ovarian cancer.

Most women with epithelial ovarian cancer (EOC) suffer from peritoneal carcinomatosis upon first clinical presentation. Extensive peritoneal carcinomatosis has a poor prognosis and its pathophysiology is not well understood. Although treatment with systemic intravenous chemotherapy is often initially successful, peritoneal recurrences occur regularly. We hypothesized that insufficient or poorly-perfused microvasculature may impair the therapeutic efficacy of systemic intravenous chemotherapy but may also limit expansive and invasive growth characteristic of peritoneal EOC metastases. In 23 patients with advanced EOC or suspicion thereof, we determined the angioarchitecture and perfusion of the microvasculature in peritoneum and in peritoneal metastases using incident dark field (IDF) imaging. Additionally, we performed immunohistochemical analysis and 3-dimensional (3D) whole tumor imaging using light sheet fluorescence microscopy of IDF-imaged tissue sites. In all metastases, microvasculature was present but the angioarchitecture was chaotic and the vessel density and perfusion of vessels was significantly lower than in unaffected peritoneum. Immunohistochemical analysis showed expression of vascular endothelial growth factor and hypoxia inducible factor 1α, and 3D imaging demonstrated vascular continuity between metastases and the vascular network of the peritoneum beneath the elastic lamina of the peritoneum. We conclude that perfusion of the microvasculature within metastases is limited, which may cause hypoxia, affect the behavior of EOC metastases on the peritoneum and limit the response of EOC metastases to systemic treatment.

Quantitative assessment of liver function using hepatobiliary scintigraphy: the effect of microcirculatory alterations after portal vein embolization.

OBJECTIVES: Hepatobiliary scintigraphy using technetium-99m mebrofenin has been validated as a quantitative liver function test. Preoperative portal vein embolization (PVE) is performed in patients to increase future remnant liver function and volume. Changes in hepatic microcirculation after PVE remain largely unknown and may influence the uptake of mebrofenin. The aim was to evaluate microcirculatory changes after PVE to examine differences in perfusion that might influence the uptake of mebrofenin, and consequently, assessment of function. PATIENTS AND METHODS: Patients undergoing liver resection with or without preoperative PVE were included. Future remnant liver volume and function were measured before and after PVE. Hepatic microcirculation was measured in the embolized and the nonembolized lobes during resection. Microcirculatory flow index, perfused vessel density, sinusoidal diameter and red blood cell velocity were assessed. RESULTS: A total of 16 patients, eight with preoperative PVE and eight control patients without PVE, were included. After PVE, both function and volume of the nonembolized lobe were significantly increased, and the functional increase exceeded the increase in volume. Perfused vessel density and sinusoidal diameter were significantly higher in the nonembolized liver lobe (P<0.002 and <0.04). No significant differences between both lobes were found concerning microcirculatory flow index or red blood cell velocity. CONCLUSION: After PVE, the nonembolized lobe had a significantly higher (functional) microvascular density compared with the embolized lobe, without differences in microvascular flow. These findings indicate that the measured functional increase using hepatobiliary scintigraphy, which exceeded the volumetric increase, was not the consequence of an increase in hepatic perfusion, therefore, providing adequate representation of the liver function.

Identification and quantification of human microcirculatory leukocytes using handheld video microscopes at the bedside.

Leukocyte recruitment and adhesion to the endothelium are hallmarks of systemic inflammation that manifest in a wide range of diseases. At present, no method is available to directly measure leukocyte kinetics at the bedside. In this study, we validate a new method to identify and quantify microcirculatory leukocytes observed by handheld vital microscopy (HVM) using space-time diagram (STD) analysis. Video clips ( n = 59) containing one capillary-postcapillary venule unit where leukocytes could be observed emanating from a capillary into a venule in cardiac surgery patients ( n = 20) were included. STD analysis and manual counting were used to quantify the number of leukocytes (total, rolling, and nonrolling). Pearson's correlation and Bland-Altman analysis were used to determine agreement between the STDs and manual counting. For reproducibility, intra- and interobserver coefficients of variation (CVs) were assessed. Leukocyte (rolling and nonrolling) and red blood cell velocities were assessed. The STDs and manual counting procedures for the quantification of rolling leukocytes showed good agreement ( r = 0.8197, P < 0.0001), with a Bland-Altman analysis mean difference of -0.0 (-6.56; 6.56). The overall intraobserver CV for the STD method was 1.5%. The overall interobserver CVs for the STD and the manual method were 5.6% and 9.4%, respectively. The nonrolling velocity was significantly higher than the rolling velocity (812 ± 519 µm/s vs. 201 ± 149 µm/s, P = 0.001). STD results agreed with the manual counting procedure results, had a better reproducibility, and could assess the leukocyte velocity. STD analysis using bedside HVM imaging presented a new methodology for quantifying leukocyte kinetics and functions in the microcirculation. NEW & NOTEWORTHY In this study, we introduce space-time diagram analysis of sublingual microcirculation imaging using handheld vital microscopy to identify and quantify the presence and kinetics of human microcirculatory leukocytes. We validated the methodology by choosing anatomical units consisting of a capillary connected to a venule, which allowed precise identification of leukocytes.