The effect of CO2 laser therapy on vaginal microcirculatory parameters in an animal model for genitourinary syndrome of menopause.

BACKGROUND: Vaginal laser therapy for the treatment of genitourinary syndrome of menopause (GSM) has been introduced to the market with limited (pre)clinical and experimental evidence supporting its efficacy. It is suggested that vaginal laser therapy increases epithelial thickness and improves vascularization, but the underlying biological working mechanism has not been substantiated yet. OBJECTIVE: To evaluate the effects of CO2 laser therapy on vaginal atrophy using noninvasive incident dark field (IDF) imaging in a large animal model for GSM. DESIGN, SETTING, AND PARTICIPANTS: An animal study was conducted between 2018 and 2019 and included 25 Dohne Merino ewes, of which 20 underwent bilateral ovariectomy (OVX) to induce iatrogenic menopause, and 5 did not. The total study duration was 10 months. INTERVENTIONS: Five months after OVX, ovariectomized ewes received monthly applications of CO2 laser (n = 7), vaginal estrogen (n = 7), or no treatment (n = 6) for 3 months. IDF imaging was performed monthly in all animals. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was the proportion of image sequences containing capillary loops (angioarchitecture). Secondary outcomes included focal depth (epithelial thickness), and quantitative measures of vessel density and perfusion. Treatment effects were evaluated using ANCOVA and binary logistic regression. RESULTS AND LIMITATIONS: Compared to OVX-only, ewes treated with estrogen demonstrated a higher capillary loops proportion (4% vs. 75%, p < 0.01), and higher focal depth (60 (IQR 60-80) vs. 80 (IQR 80-80) p < 0.05). CO2 laser therapy did not change microcirculatory parameters. As the ewes' vaginal epithelium is thinner than that of humans, it may demand different laser settings. CONCLUSIONS: In a large animal model for GSM, CO2 laser therapy does not affect microcirculatory outcomes related to GSM, whereas vaginal estrogen treatment does. Until more homogeneous and objective evidence about its efficacy is available, CO2 laser therapy should not be adopted into widespread practice for treating GSM.

Intra-renal microcirculatory alterations on non-traumatic hemorrhagic shock induced acute kidney injury in pigs.

Acute kidney injury (AKI) is frequently seen in patients with hemorrhagic shock due to hypotension, tissue hypoxia, and inflammation despite adequate resuscitation. There is a lack of information concerning the alteration of renal microcirculation and perfusion during shock and resuscitation. The aim of this study was to investigate the possible role of renal microcirculatory alterations on development of renal dysfunction in a pig model of non-traumatic hemorrhagic shock (HS) induced AKI.Fully instrumented female pigs were divided into the two groups as Control (n = 6) and HS (n = 11). HS was achieved by withdrawing blood until mean arterial pressure (MAP) reached around 50 mmHg. After an hour cessation period, fluid resuscitation with balanced crystalloid was started for the duration of 1 h. The systemic and renal hemodynamics, renal microcirculatory perfusion (contrast-enhanced ultrasound (CEUS)) and the sublingual microcirculation were measured.CEUS peak enhancement was significantly increased in HS during shock, early-, and late resuscitation indicating perfusion defects in the renal cortex (p < 0.05 vs. baseline, BL) despite a stable renal blood flow (RBF) and urine output. Following normalization of systemic hemodynamics, we observed persistent hypoxia (high lactate) and high red blood cell (RBC) velocity just after initiation of resuscitation resulting in further endothelial and renal damage as shown by increased plasma sialic acid (p < 0.05 vs. BL) and NGAL levels. We also showed that total vessel density (TVD) and functional capillary density (FCD) were depleted during resuscitation (p < 0.05).In this study, we showed that the correction of systemic hemodynamic variables may not be accompanied with the improvement of renal cortical perfusion, intra-renal blood volume and renal damage following fluid resuscitation. We suggest that the measurement of renal injury biomarkers, systemic and renal microcirculation can be used for guiding to the optimization of fluid therapies.

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.

Hydroxyl Ethyl Starch (HES) Preserves Intrarenal Microcirculatory Perfusion Shown by Contrast-Enhanced Ultrasound (Ceus), and Renal Function in a Severe Hemodilution Model in Pigs.

ABSTRACT: Acute normovolemic hemodilution (ANH) is associated with low oxygen carrying capacity of blood and purposed to cause renal injury in perioperative setting. It is best accomplished in a perioperative setting by a colloid such as hydroxyl ethyl starch (HES) due its capacity to fill the vascular compartment and maintain colloidal pressure. However, alterations of intra renal microvascular perfusion, flow and its effects on renal function and damage during ANH has not been sufficiently clarified. Based on the extensive use of HES in the perioperative setting we tested the hypothesis that the use of HES during ANH is able to perfuse the kidney microcirculation adequately without causing renal dysfunction and injury in pigs. Hemodilution (n = 8) was performed by stepwise replacing blood with HES to hematocrit (Hct) levels of 20% (T1), 15% (T2), and 10% (T3). Seven control animals were investigated. Systemic and renal hemodynamics were monitored. Renal microcirculatory perfusion was visualized and quantified using contrast-enhanced ultrasound (CEUS) and laser speckle imaging (LSI). In addition, sublingual microcirculation was measured by handheld vital microscopy (HVM). Intrarenal mean transit time of ultrasound contrast agent (IRMTT-CEUS) was reduced in the renal cortex at Hct 10% in comparison to control at T3 (1.4 ±â€Š0.6 vs. 2.2 ±â€Š0.7 seconds, respectively, P < 0.05). Although renal function was preserved, the serum neutrophil gelatinase-associated lipocalin (NGAL) levels was higher at Hct 10% (0.033 ±â€Š0.004 pg/µg protein) in comparison to control at T3 (0.021 ±â€Š0.002 pg/µg protein. A mild correlation between CO and IRMTT (renal RBC velocity) (r -0.53; P = 0.001) and CO and NGAL levels (r 0.66; P = 0.001) was also found. Our results show that HES induced ANH is associated with a preserved intra renal blood volume, perfusion, and function in the clinical range of Hct (<15%). However, at severely low Hct (10%) ANH was associated with renal injury as indicated by increased NGAL levels. Changes in renal microcirculatory flow (CEUS and LSI) followed those seen in the sublingual microcirculation measured with HVM.

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.

Resuscitation with PEGylated carboxyhemoglobin preserves renal cortical oxygenation and improves skeletal muscle microcirculatory flow during endotoxemia.

PEGylated carboxyhemoglobin (PEGHbCO), which has carbon monoxide-releasing properties and plasma expansion and oxygen-carrying properties, may improve both skeletal microcirculatory flow and renal cortical microcirculatory Po2 (CµPo2) and, subsequently, limit endotoxemia-induced acute kidney injury. Anesthetized, ventilated Wistar albino rats (n = 44) underwent endotoxemic shock. CµPo2 was measured in exposed kidneys using a phosphorescence-quenching method. Rats were randomly assigned to the following five groups: 1) unresuscitated lipopolysaccharide (LPS), 2) LPS + Ringer's acetate (RA), 3) LPS + RA + 0.5 µg·kg·-1min-1 norepinephrine (NE), 4) LPS + RA + 320 mg/kg PEGHbCO, and 5) LPS + RA + PEGHbCO + NE. The total volume was 30 mL/kg in each group. A time control animal group was used. Skeletal muscle microcirculation was assessed by handheld intravital microscopy. Kidney immunohistochemistry and myeloperoxidase-stained leukocytes in glomerular and peritubular areas were analyzed. Endotoxemia-induced histological damage was assessed. Plasma levels of IL-6, heme oxygenase-1, malondialdehyde, and syndecan-1 were assessed by ELISA. CµPo2 was higher in the LPS + RA + PEGHbCO-resuscitated group, at 35 ± 6mmHg compared with 21 ± 12 mmHg for the LPS+RA group [mean difference: -13.53, 95% confidence interval: (-26.35; -0.7156), P = 0.035]. The number of nonflowing, intermittent, or sluggish capillaries was smaller in groups infused with PEGHbCO compared with RA alone (P < 0.05), while the number of normally perfused vessels was greater (P < 0.05). The addition of NE did not further improve CµPo2 or microcirculatory parameters. Endotoxemia-induced kidney immunohistochemistry and histological alterations were not mitigated by PEGHbCO 1 h after resuscitation. Renal leukocyte infiltration and plasma levels of biomarkers were similar across groups. PEGHbCO enhanced CµPo2 while restoring skeletal muscle microcirculatory flow in previously nonflowing capillaries. PEGHbCO should be further evaluated as a resuscitation fluid in mid- to long-term models of sepsis-induced acute kidney injury.

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.

Site-specific pharmaco-laser therapy: A novel treatment modality for refractory port wine stains.

Despite extensive efforts to optimize laser therapy, i.e., the current gold standard treatment, a majority of port wine stain (PWS) patients responds suboptimally to laser therapy. This paper describes the niceties of a novel PWS treatment modality termed site-specific pharmaco-laser therapy (SSPLT). In contrast to the classic approach of enhancing the extent of intravascular photocoagulation (the photothermal response), SSPLT focuses on optimization of post-irradiation thrombus formation (i.e., the hemodynamic response) by combining conventional laser therapy with the administration of thermosensitive drug delivery systems that encapsulate prothrombotic and antifibrinolytic drugs. The aim of SSPLT is to instill complete lumenal occlusion in target vessels, which has been linked to optimal PWS blanching. RELEVANCE FOR PATIENTS: The current treatment options for PWS patients are limited in efficacy. Novel therapeutic modalities are needed to more effectively treat patients with recalcitrant PWSs. SSPLT is an experimental-stage treatment modality that could serve as an adjuvant to pulsed dye laser therapy for a selected group of patients whose PWS is ill-responsive to standard treatment. The expected clinical result of SSPLT is improved lesional blanching.

MicroTools enables automated quantification of capillary density and red blood cell velocity in handheld vital microscopy.

Direct assessment of capillary perfusion has been prioritized in hemodynamic management of critically ill patients in addition to optimizing blood flow on the global scale. Sublingual handheld vital microscopy has enabled online acquisition of moving image sequences of the microcirculation, including the flow of individual red blood cells in the capillary network. However, due to inherent content complexity, manual image sequence analysis remained gold standard, introducing inter-observer variability and precluding real-time image analysis for clinical therapy guidance. Here we introduce an advanced computer vision algorithm for instantaneous analysis and quantification of morphometric and kinetic information related to capillary blood flow in the sublingual microcirculation. We evaluated this technique in a porcine model of septic shock and resuscitation and cardiac surgery patients. This development is of high clinical relevance because it enables implementation of point-of-care goal-directed resuscitation procedures based on correction of microcirculatory perfusion in critically ill and perioperative patients.

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.

Recruitment of sublingual microcirculation using handheld incident dark field imaging as a routine measurement tool during the postoperative de-escalation phase-a pilot study in post ICU cardiac surgery patients.

BACKGROUND: Management of tissue perfusion following cardiac surgery is a challenging task where common clinical parameters do not reflect microcirculatory dysfunction. Heterogeneity in blood flow perfusion and abnormalities in capillary density characterize microcirculatory dysfunction. The restoration of a normal microcirculation may become a novel target for therapy in the future in addition to macrocirculatory parameters. The aim of this study is to determine how the sublingual microcirculatory parameters vary at the bedside in post-cardiac surgery patients which underwent diuretic therapy to correct fluid overload. METHODS: In this prospective observational pilot study, video clips of sublingual microcirculation in post-cardiac surgery patients receiving furosemide and/or spironolactone to achieve normal fluid balance were recorded using Cytocam-IDF imaging. Data was obtained on the first (T0), second (T1), and third (T2) day after the patients left the intensive care unit (ICU). Measurements were analyzed off-line to obtain the following microcirculatory parameters: total vessel density (TVD), microcirculatory flow index (MFI), proportion of perfused vessel (PPV), and perfused vessel density (PVD). Macrocirculatory parameters and body weight were also collected at these time points. RESULTS: Ninety measurements were performed in ten post ICU cardiac surgery patients. Thirteen measurements were excluded due to quality reasons; these excluded measurements were spread across the patients and time points, and there was no loss of patients or time points. An increase in TVD was observed from T0 to T1 (20 ± 2.7 to 24 ± 3.2 mm/mm2; p = 0.0410) and from T0 to T2 (20 ± 2.7 to 26 ± 3.3 mm/mm2; p = 0.0005). An increase in PVD was present from T0 to T1 (19 ± 2.3 to 24 ± 3.5 mm/mm2; p = 0.0072) and from T0 to T2 (19 ± 2.3 to 26 ± 3.4 mm/mm2, p = 0.0008). Fluid overload was assessed through a positive cumulative fluid balance on the day of ICU discharge. CONCLUSIONS: Cytocam-IDF imaging to monitor microcirculation as a daily parameter is feasible and could become a valuable tool to non-invasively assess the tissue oxygenation at the bedside. An increase in TVD and PVD (functional capillary density) indicated the recruitment of the sublingual microcirculation in patients with diuretic therapy. Future research is needed to prove the correlation between the recruitment of the sublingual microcirculation and the de-escalation phase of the fluid management.

Dynamic Contrast-Enhanced Ultrasound Identifies Microcirculatory Alterations in Sepsis-Induced Acute Kidney Injury.

OBJECTIVES: We developed quantitative methods to analyze microbubble kinetics based on renal contrast-enhanced ultrasound imaging combined with measurements of sublingual microcirculation on a fixed area to quantify early microvascular alterations in sepsis-induced acute kidney injury. DESIGN: Prospective controlled animal experiment study. SETTING: Hospital-affiliated animal research institution. SUBJECTS: Fifteen female pigs. INTERVENTIONS: The animals were instrumented with a renal artery flow probe after surgically exposing the kidney. Nine animals were given IV infusion of lipopolysaccharide to induce septic shock, and six were used as controls. MEASUREMENTS AND MAIN RESULTS: Contrast-enhanced ultrasound imaging was performed on the kidney before, during, and after having induced shock. Sublingual microcirculation was measured continuously using the Cytocam on the same spot. Contrast-enhanced ultrasound effectively allowed us to develop new analytical methods to measure dynamic variations in renal microvascular perfusion during shock and resuscitation. Renal microvascular hypoperfusion was quantified by decreased peak enhancement and an increased ratio of the final plateau intensity to peak enhancement. Reduced intrarenal blood flow could be estimated by measuring the microbubble transit times between the interlobar arteries and capillary vessels in the renal cortex. Sublingual microcirculation measured using the Cytocam in a fixed area showed decreased functional capillary density associated with plugged sublingual capillary vessels that persisted during and after fluid resuscitation. CONCLUSIONS: In our lipopolysaccharide model, with resuscitation targeted at blood pressure, contrast-enhanced ultrasound imaging can identify renal microvascular alterations by showing prolonged contrast enhancement in microcirculation during shock, worsened by resuscitation with fluids. Concomitant analysis of sublingual microcirculation mirrored those observed in the renal microcirculation.

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.

A LED-based phosphorimeter for measurement of microcirculatory oxygen pressure.

Quantitative measurements of microcirculatory and tissue oxygenation are of prime importance in experimental research. The noninvasive phosphorescence quenching method has given further insight into the fundamental mechanisms of oxygen transport to healthy tissues and in models of disease. Phosphorimeters are devices dedicated to the study of phosphorescence quenching. The experimental applications of phosphorimeters range from measuring a specific oxygen partial pressure (Po2) in cellular organelles such as mitochondria, finding values of Po2 distributed over an organ or capillaries, to measuring microcirculatory Po2 changes simultaneously in several organ systems. Most of the current phosphorimeters use flash lamps as a light excitation source. However, a major drawback of flash lamps is their inherent plasma glow that persists for tens of microseconds after the primary discharge. This complex distributed excitation pattern generated by the flash lamp can lead to inaccurate Po2 readings unless a deconvolution analysis is performed. Using light-emitting diode (LED), a rectangular shaped light pulse can be generated that provides a more uniformly distributed excitation signal. This study presents the design and calibration process of an LED-based phosphorimeter (LED-P). The in vitro calibration of the LED-P using palladium(II)-meso-tetra(4-carboxyphenyl)-porphyrin (Pd-TCCP) as a phosphorescent dye is presented. The pH and temperature were altered to assess whether the decay times of the Pd-TCCP measured by the LED-P were significantly influenced. An in vivo validation experiment was undertaken to measure renal cortical Po2 in a rat subjected to hypoxic ventilation conditions and ischemia/reperfusion. The benefits of using LEDs as a light excitation source are presented.