Intravital Videomicroscopy Research Articles

Conditions that promote transcellular neutrophil migration in vivo

Circulating leukocytes enter tissue either through endothelial junctions (paracellular) or via a pore through the body of endothelial cells (transcellular). We have previously shown that genetically replacing VE-cadherin with a VE-cadherin-α-catenin (VEC-αC) fusion construct—which binds constitutively to actin—obstructs junctions, and blocks leukocyte extravasation in lung, skin and postcapillary venules of cremaster muscle. However, neutrophil recruitment into the inflamed peritoneal cavity was unimpaired. Investigating reasons for this, here, we visualized neutrophil diapedesis by 3D intravital video microscopy in the cremaster muscle and omentum, the major site of neutrophil recruitment into the peritoneal cavity. We found that 80% of neutrophil-extravasation occurred through HEVs in the omentum, which was unimpaired by VEC-αC. In addition, in larger venules (60–85 µm) of both tissues, less than 15% of neutrophils extravasated transcellularly in WT mice. However, in VEC-α-C mice, transcellular diapedesis increased severalfold in the omentum, but not in the cremaster. In line with this, omental venules expressed higher levels of ICAM-1 and atypical chemokine receptor 1. Furthermore, only in the omentum, VEC-αC expression caused reduced elongation of venular endothelium in flow-direction, suggesting different biomechanical properties. Collectively, VEC-αC does not inhibit paracellular transmigration in all types of venules and can modulate the diapedesis route.

Microcirculation-driven mitochondrion dysfunction during the progression of experimental sepsis

Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of microcirculatory and mitochondrial functions in a rodent model of intraabdominal sepsis. Fecal peritonitis was induced in rats, and multi-organ failure (MOF) was evaluated 12, 16, 20, 24 or 28 h later (n = 8/group, each) using rat-specific organ failure assessment (ROFA) scores. Ileal microcirculation (proportion of perfused microvessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI)) was monitored by intravital video microscopy, and mitochondrial respiration (OxPhos) and outer membrane (mtOM) damage were measured with high-resolution respirometry. MOF progression was evidenced by increased ROFA scores; microcirculatory parameters followed a parallel time course from the 16th to 28th h. Mitochondrial dysfunction commenced with a 4-h time lag with signs of mtOM damage, which correlated significantly with PPV, while no correlation was found between HI and OxPhos. High diagnostic value was demonstrated for PPV, mtOM damage and lactate levels for predicting MOF. Our findings indicate insufficient splanchnic microcirculation to be a possible predictor for MOF that develops before the start of mitochondrial dysfunction. The adequate subcellular compensatory capacity suggests the presence of mitochondrial subpopulations with differing sensitivity to septic insults.

Thrombin-activatable fibrinolysis inhibitor and sex modulate thrombus stability and pulmonary embolism burden in a murine model

BackgroundThrombin-activatable fibrinolysis inhibitor (TAFI) levels are positively correlated with the risk of thrombosis. The mechanism of how TAFI affects venous thromboembolism (VTE) remains uncertain. In addition, the role of sex on the risk of VTE has also been studied. However, their association also remains unclear. ObjectivesTo investigate how TAFI and/or sex affect venous thrombus stability and consequent pulmonary embolism (PE). MethodsFerric chloride-induced thrombi were formed within the femoral veins of male and female wild-type (WT) or TAFI-knockout (Cpb2-/-) mice. Thrombi were imaged over 2 hours using intravital videomicroscopy to quantify embolization and thrombus size over time. Lungs were examined by immunohistochemistry to quantify (a) emboli and (b) fibrin composition of these emboli. ResultsEmbolization events in female mice were higher than in males (7.9-fold in WT and 3.1-fold in Cpb2-/- mice). Although the maximal thrombus sizes were not different across groups, Cpb2-/- mice had thrombi that were, on average, 24% smaller at the end of the 2-hour experiment than WT mice. Loss of TAFI led to a 4.0- and 2.8-fold increase in PE burden in males and females, respectively, while sex had no influence. Pulmonary emboli in Cpb2-/- mice had higher fibrin composition compared with WT mice. ConclusionFemale mice had less stable venous thrombi than male mice, suggesting a higher risk of PE in females with deep vein thrombosis. Mice lacking TAFI had more thrombus degradation and higher PE burden than WT mice. These results confirm the role of TAFI in venous thrombosis.

Conjugation with Tris Decreases the Risk of Ketoprofen-Induced Mucosal Damage and Reduces Inflammation-Associated Methane Production in a Rat Model of Colitis.

We have designed a new compound from the non-steroidal anti-inflammatory drug (NSAID) ketoprofen (Ket) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) precursors, with the aim to reduce the gastrointestinal (GI) side effects of NSAID therapies. We investigated mucosal reactions in a standard rat model of colitis together with methane generation as a possible indicator of pro-inflammatory activation under this condition (approval number: V./148/2013). Whole-body methane production (photoacoustic spectroscopy) and serosal microcirculation (intravital videomicroscopy) were measured, and mucosal damage was assessed (conventional histology; in vivo laser-scanning endomicroscopy). Inflammatory markers were measured from tissue and blood samples. Colitis induced an inflammatory response, morphological colonic damage and increased methane output. Ket treatment lowered inflammatory activation and colonic mucosal injury, but macroscopic gastric bleeding and increased methane output were present. Ket-Tris reduced inflammatory activation, methane emission and colonic mucosal damage, without inducing gastric injury. Conjugation with Tris reduces the GI side effects of Ket and still decreases the inflammatory response in experimental colitis. Methane output correlates with the mucosal inflammatory response and non-invasively demonstrates the effects of anti-inflammatory treatments.

Fixing skeletal muscle PO2 eliminates hyperinsulinemic microvascular blood flow response.

To determine if insulin-mediated hyperemia is partially dependent on local muscle oxygen concentration. Sprague-Dawley rats were anesthetized, and the extensor digitorum longus (EDL) was reflected onto an inverted microscope. Intravital video microscopy sequences were recorded during baseline and hyperinsulinemic euglycemia. The muscle was reflected over a glass stage insert (Experiment 1a and 1b), or over a gas exchange chamber (Experiment 2), and microvascular capillary blood flow was recorded during sequential changes (7%-12%-2%-7%) of oxygen (O2 ) concentration. Blood flow was measured by the red blood cell supply rate (SR) in number of cells per second. All animal protocols were approved by Memorial University's Institutional Animal Care Committee. In Experiment 1a, SR increased from 8.0 to 14.0 cells/s at baseline to euglycemia (p=.01), while no significant SR variation was detected after performing a sham hyperinsulinemic euglycemic clamp (Experiment 1b). In Experiment 2, SR decreased at 12% O2 and increased at 2% O2 , compared to 7% O2 , under both experimental conditions. Magnitude of SR responses to oxygen oscillations during euglycemia were not different to those at baseline at each O2 concentration (p > .9). Our results suggest that increased blood flow observed in response to insulin is eliminated if tissue oxygen microenvironment is fixed at a given oxygen concentration.

Conditional over-expression of heme-oxygenase 1 in myelomonocytic cells reduces hypertension and vascular inflammation

Abstract Background Arterial hypertension is one of the most important modifiable risk factors for all-cause morbidity and mortality worldwide. Angiotensin II (Ang II) plays a pathogenic role in the development of hypertension, vascular dysfunction, inflammation and tissue damage. In the context of hypertension, heme-oxygenase 1 (HO-1) gene expression is upregulated as an antioxidant defense system in response to AngII through its action on heme catabolism, which generates carbon monoxide (CO), ferritin and biliverdin, which is reduced to bilirubin by biliverdin reductase A (BLVRA). Previous studies have shown the important role of HO-1 in the maturation and migration of immune cells. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion damage. However, the role of myeloid cell specific HO-1 in the detrimental effects of AngII induced vascular dysfunction has not yet been explored. Objectives To investigate the potential vascular protection of myeloid cell specific overexpression of HO-1 in AngII-induced arterial hypertension. Methods Hypertension was induced in 8–13 weeks old male mice with selective over-expression of HO-1 (HO-1indLysMcre) in myelomonocytic cells versus LysMCre/wt by AngII infusion (1mg/kg/d). Blood pressure was recorded by tail-cuff. Bilirubin levels were quantified in plasma by High Performance Liquid Chromatography (HPLC). The quantification of adherent and rolling leukocytes in carotid arteries was detected by intravital video microscopy (IVM). Endogenuos Thrombin potential (ETP) was measured in platelet rich plasma (PRP) and platelets poor plasma (PPP) by Calibrated Automated Thrombogram (CAT) assay. Endothelium-dependent vasodilation was assessed in isolated aortic rings by concentration-relaxation curves in response to acetylcholine (ACh). Results AngII-infused HO-1indLysMcre had decreased blood pressure values and improved endothelial function as compared to LysMcre controls. IVM revealed reduced leukocyte rolling and adhesion to the vascular endothelium in AngII infused HO-1indLysMcre mice compared to controls, paralleled by reduced expression of NOX-2 mediated oxidative stress and vascular inflammation in AngII-induced arterial hypertension by decreasing VCAM-1, CCR2 and MCP-1 expression. mRNA analysis revealed an increased expression of BLVRA in liver, spleen, heart and aortic tissues in response to AngII, which was higher in HO-1indLysMcre mice than in controls. By CAT assay we registered a decrease ETP in PPP and PRP in HO-1ind mice infused with AngII compatible with less abundance of inflammatory platelets, potentially regulated by BLVRA activity. This was supported by increased in bilirubin levels in response to AngII, which was higher in HO-1indLysMcre mice than in controls. Conclusion Myeloid cell specific overexpression of HO-1 confers anti-inflammatory protection to the vasculature in AngII induced hypertension. This effect is, at least in part, mediated by BLVRA. Funding Acknowledgement Type of funding sources: None.

The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease

AbstractSickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the β-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Quantifying Longitudinal Network Geometry Alterations in Rat Skeletal Microvasculature Subject to Ischemia

Study objectiveIntra‐vital video microscopy (IVVM) data of rat subcutaneous skeletal muscle microvasculature under ischemic conditions at two time points (0 hours, 3 hours) was obtained from our collaborator. The objectives of this study are to elicit network geometries from the IVVM data and quantify the alterations in the network statistics of the geometries due to ischemia.HypothesisWe hypothesize that ischemia causes blood vessel de‐recruitment.MethodsIVVM data from six experiments was obtained, where each experiment provided a video at the start (0 hours) and end (3 hours) of the experiment. Using consecutive still images from the videos, vessel centerlines were manually drawn using Procreate and the NIH software, ImageJ. The included vessels were chosen based on two features: (1) visible red blood cell flow and (2) clearly defined edges. The centerlines were used to guide thresholding by identifying appropriate vessels. This was followed by the generation of binarized representations of the microvascular networks. The network statistics were quantified by measuring vessel segment lengths and diameters, counting the number of bifurcations, and calculating vessel density in ImageJ.ResultsAt 3 hours, the average vessel length changed from 283.91 + 252.14 pixels to 316.75 + 292.37 pixels, the average diameter changed from 16.02 + 7.99 pixels to 15.42 + 7.50 pixels, the average number of bifurcations changed from 32.42 to 18.92 and the average vessel density changed from 0.0085 + 0.0017 to 0.0061 + 0.0024.ConclusionThere is a clear decrease in average diameter, vessel density and the number of bifurcations characterizing the microvascular networks. However, an increase in the average vessel length accompanies these findings. The data indicates that ischemia may cause de‐recruitment of blood vessels.

Clamping Skeletal Muscle PO2 Eliminates Hyperinsulinemic Microvascular Blood Flow Response

ObjectiveTo determine if insulin mediated hyperemia is partially dependent on local muscle oxygen concentration.HypothesisThe blood flow responses to insulin are partially mediated by the local metabolic demand reflecting the increased oxygen consumption required to uptake and convert glucose into glycogen in skeletal muscle.Methods16 male Sprague Dawley rats (161g ‐ 204g) were anesthetized with sodium pentobarbital, the carotid artery and jugular vein were then cannulated for blood pressure monitoring and fluid resuscitation. Following cannulation, the extensor digitorum longus (EDL) was isolated and reflected onto an inverted microscope. Intravital video microscopy sequences of the EDL microcirculation were recorded during baseline and following hyperinsulinemic euglycemia or sham in all experiments. The muscle was reflected over a glass stage insert during Experiment 1a and 1b which allowed for direct observation of microvascular blood flow response to systemic insulin infusion in Experiment 1a. Experiment 1b consisted of a sham hyperinsulinemic euglycemic clamp which involved infusing saline at similar rates as insulin and glucose in standard clamp procedures. In Experiment 2, the EDL was reflected over a gas exchange chamber and microvascular capillary blood flow was recorded during sequential changes (7%‐12%‐2%‐7%) of oxygen concentration [O2] within the chamber during baseline and euglycemic conditions. The gas exchange chamber is fitted into the stage of an inverted microscope that interfaces directly with the EDL via a gas permeable polydimethylsiloxane membrane. By adjusting the concentration of gases flowing through the gas exchange chamber we have direct control of the [O2] within the overlying muscle. Hemodynamic measurements including capillary red blood cell (RBC) velocity, supply rate (SR), hematocrit, and oxygen saturation (SO2) were taken from in focus capillaries in the EDL during each experiment and analyzed offline using custom MATLAB software. All animal protocols were approved by Memorial University’s Institutional Animal Care Committee.ResultsIn Experiment 1a, the hyperinsulinemic euglycemic clamp caused a significant increase in SR from 8.9 ± 3.1 to 14.2 ± 4.9 cells/s at baseline to euglycemia (p = 0.01; N = 6, 677 capillaries at baseline and 552 at clamp), while no significant SR variation was detected after performing a sham clamp in Experiment 1b (11.0 ± 1.6 cells/sec at baseline and 11.4 ± 3.7 cells/sec at sham clamp; N = 6, 837 capillaries at baseline and 724 at sham clamp). In Experiment 2 using the gas exchange chamber, SR significantly decreased at 12% O2 (p = 0.01, p < 0.03) and significantly increased at 2% O2 (p = 0.01, p < 0.04), compared to baseline 7% O2, under both baseline and euglycemic conditions respectively (N = 6, 313 capillaries at baseline and 195 at clamp). SR responses to oxygen oscillations during euglycemia were not different to those at baseline for each O2 concentration (p > 0.9). SO2 significantly increased at 12% O2 and significantly decreased at 2% O2 under both baseline and euglycemic conditions (p < 0.05).ConclusionsOur saturation measurements demonstrate the effectiveness of using a gas exchange device to manipulate local oxygen concentrations in the muscle. Our SR results suggest the increase in blood flow observed in response to insulin is eliminated if the tissue oxygen microenvironment is fixed at a given [O2].

Microvascular Blood Flow Response to Hyperinsulinemic‐Euglycemic Clamp in Zucker Diabetic Sprague Dawley Rat Model of Type 2 Diabetes

ObjectiveQuantify and characterize the skeletal muscle microvascular blood flow in Zucker Diabetic Sprague Dawley (ZDSD) rat model of T2D at rest and during a hyperinsulinemic‐euglycemic clamp.HypothesisChronic hyperglycemia in the ZDSD rat model of T2D causes blunted capillary hemodynamic response to hyperinsulinemic‐euglycemic clamp compared to the age and diet matched control Sprague Dawley (SD) rats.MethodsBiweekly blood glucose and conscious blood pressure measurements were collected from 7‐ to 25‐week‐old male SD (n=12) and ZDSD (n=18) rats. At 27 weeks of age, animals were fasted overnight prior to intravital video microscopy experiments. On the day of experiment animals were anaesthetized with an intraperitoneal injection of pentobarbital, tracheotomized, and carotid and external jugular cannulas were inserted to monitor physiological status and for administration of fluids. The extensor digitorum longus (EDL) muscle of the lower hind limb was isolated, blunt dissected and reflected over a glass coverslip on the stage of an inverted microscope. Recordings of the EDL were completed at baseline and during hyperinsulinemic‐euglycemic clamp. Hyperinsulinemic‐euglycemic clamp was achieved by continuous intravenous infusion of insulin (Humulin R 2U/hr/kg) and a 50% glucose mixture to titrate blood glucose to euglycemia (5‐7 mM). Mixed blood samples were collected from a tail clip to determine systemic blood glucose. Analysis of intravital videos were completed offline using custom MATLAB software. ZDSD rats that had fasting blood glucose of less than 20 mM at 19 weeks of age were excluded from the hyperglycemic group. Animals exhibiting aberrant microvascular blood flow at baseline or during euglycemic clamp were excluded from the data set. All animal protocols were approved by Memorial University of Newfoundland’s Animal Care and Use Committee.ResultsHyperglycemic ZDSD rats had a significantly higher blood glucose than SD controls from 17 to 25 weeks old and at the beginning of euglycemic clamp (26.13 ± 0.97 and 5.95 ± 0.37, p < 0.0001, Fig 1A). SD rats had a significantly higher glucose infusion rate than ZDSD rats (22.58 ± 1.81 and 13.19 ± 2.76, p < 0.0001, Fig 1B). SD rats had a significant increase in red blood cell (RBC) velocity (149.8 ± 56.71 and 210.0 ± 62.14 µm/s, p = 0.0051, Fig 2A), RBC supply rate (SR) (12.49 ± 8.23 and 19.91 ± 12.25 cells/s, p = 0.004, Fig 2B) and RBC oxygen saturation(sO2) (38.05 ± 11.65 and 45.70 ± 10.13, p = 0.014, Fig 2D) between baseline and euglycemia. However, ZDSD animals had no significant difference in RBC velocity (178.1 ± 63.91 and 194.7 ± 37.69 µm/s, p = 0.3683, Fig 2A), RBC supply rate (14.63 ± 7.55 and 20.03 ± 7.46 cells/s, p = 0.0704, Fig 2B), hematocrit (31.03 ± 3.23 and 33.02 ± 6.22 %, p = 0.4309, Fig 2C), and RBC sO2 (61.91 ± 11.40 and 64.00 ± 11.59, p = 0.5324 Fig 2D). The RBC sO2 was significantly higher in ZDSD than SD at baseline (61.91 ± 11.40 compared to 38.05 ± 11.65, p = 0.0166, Fig 2D).ConclusionSD rats had a robust increase in capillary hemodynamics during hyperinsulinemic‐euglycemic clamp. However, the skeletal muscle capillary hemodynamics of ZDSD rats did not significantly change between baseline and during euglycemia. These finding suggests a potential impairment in skeletal muscle microvascular blood flow response in this type 2 diabetic rodent mode. The elevated RBC sO2 in ZDSD rats may indicate a dysregulation of capillary blood flow in the ZDSD model.

TLR4 mutation protects neurovascular function and cognitive decline in high-fat diet-fed mice

BackgroundMetabolic syndrome (MS) is defined as a low-grade proinflammatory state in which abnormal metabolic and cardiovascular factors increase the risk of developing cardiovascular disease and neuroinflammation. Events, such as the accumulation of visceral adipose tissue, increased plasma concentrations of free fatty acids, tissue hypoxia, and sympathetic hyperactivity in MS may contribute to the direct or indirect activation of Toll-like receptors (TLRs), specifically TLR4, which is thought to be a major component of this syndrome. Activation of the innate immune response via TLR4 may contribute to this state of chronic inflammation and may be related to the neuroinflammation and neurodegeneration observed in MS. In this study, we investigated the role of TLR4 in the brain microcirculation and in the cognitive performance of high-fat diet (HFD)-induced MS mice.MethodsWild-type (C3H/He) and TLR4 mutant (C3H/HeJ) mice were maintained under a normal diet (ND) or a HFD for 24 weeks. Intravital video-microscopy was used to investigate the functional capillary density, endothelial function, and endothelial–leukocyte interactions in the brain microcirculation. Plasma concentrations of monocyte chemoattractant protein-1 (MCP-1), adipokines and metabolic hormones were measured with a multiplex immunoassay. Brain postsynaptic density protein-95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the vessels, microglial activation and structural capillary density were evaluated by immunohistochemistry.ResultsThe HFD-induced MS model leads to metabolic, hemodynamic, and microcirculatory alterations, as evidenced by capillary rarefaction, increased rolling and leukocyte adhesion in postcapillary venules, endothelial dysfunction, and less coverage of astrocytes in the vessels, which are directly related to cognitive decline and neuroinflammation. The same model of MS reproduced in mice deficient for TLR4 because of a genetic mutation does not generate such changes. Furthermore, the comparison of wild-type mice fed a HFD and a normolipid diet revealed differences in inflammation in the cerebral microcirculation, possibly related to lower TLR4 activation.ConclusionsOur results demonstrate that TLR4 is involved in the microvascular dysfunction and neuroinflammation associated with HFD-induced MS and possibly has a causal role in the development of cognitive decline.

LPPR5 Expression in Glioma Affects Growth, Vascular Architecture, and Sunitinib Resistance.

Despite intensive research, glioblastoma remains almost invariably fatal. Various promising drugs targeting specific aspects of glioma biology, in addition to or as an alternative to antiproliferative chemotherapy, were not successful in larger clinical trials. Further insights into the biology of glioma and the mechanisms behind the evasive-adaptive response to targeted therapies is needed to help identify new therapeutic targets, prognostics, or predictive biomarkers. As a modulator of the canonically oncogenic Rho-GTPase pathway, Lipid phosphate phosphatase-related protein type 5 (LPPR5) is pivotal in influencing growth, angiogenesis, and therapeutic resistance. We used a GL261 murine orthotopic allograft glioma model to quantify the tumor growth and to obtain tissue for histological and molecular analysis. Epicortical intravital epi-illumination fluorescence video microscopy of the tumor cell spheroids was used to characterize the neovascular architecture and hemodynamics. GL261-glioma growth was delayed and decelerated after LPPR5 overexpression (LPPR5OE). We observed increased tumor cell apoptosis and decreased expression and secretion of vascular endothelial growth factor A in LPPR5OE glioma. Hence, an altered micro-angioarchitecture consisting of dysfunctional small blood vessels was discovered in the LPPR5OE tumors. Sunitinib therapy eliminated these vessels but had no effect on tumor growth or apoptosis. In general, LPPR5 overexpression generated a more benign, proapoptotic glioma phenotype with delayed growth and a dysfunctional vascular architecture.

Optical method to determine in vivo capillary hematocrit, hemoglobin concentration, and 3-D network geometry in skeletal muscle.

The aim of this study was to develop a tool to visualize and quantify hemodynamic information, such as hemoglobin concentration and hematocrit, within microvascular networks recorded in vivo using intravital video microscopy. Additionally, we aimed to facilitate the 3-D reconstruction of the microvascular networks. Digital images taken from an intravital video microscopy preparation of the extensor digitorum longus muscle in rats for 25 capillary segments were used. The developed algorithm was used to delineate capillaries of interest, calculate the optical density for each pixel in the image, and reconstruct the 3-D capillary geometry using the calculated light path-lengths. Subsequently, the mean corpuscular hemoglobin concentration (MCHC), hemoglobin concentration, and hematocrit for these capillaries were calculated. We evaluated the hematocrit values determined by our methodology by comparing them to those obtained using a previously published method. The hematocrit values from the proposed optical method were strongly correlated with those calculated using published methods r2 (25)=.92, p<.001, and demonstrated excellent agreement with a mean difference of 1.3% and a coefficient of variation (CV) of 11%. The average MCHC, hemoglobin concentration, and light path-lengths were 23.83g/dl, 8.06g/dl, and 3.92µm, respectively. The proposed methodology can quantify hemodynamic measurements and produce functional images for visualization of the microcirculation in vivo.

Capillary module haemodynamics and mechanisms of blood flow regulation in skeletal muscle capillary networks: Experimental and computational analysis.

The capillary module (CM), consisting of parallel capillaries from terminal arteriole to post-capillary venule, is classically considered to be the building block of complex capillary networks. In skeletal muscle, CMs form interconnected columns spanning thousands of microns, which we recently described as the capillary fascicle. However, detailed evaluation of CM haemodynamics has not been described, and may provide insight into mechanisms of blood flow regulation in the microcirculation. We used intravital videomicroscopy from resting extensor digitorum longus muscle in rats (n=9 networks, 112 capillary modules), as well as dual-phase computational modelling of blood flow in simulated CM geometries. We found that the mean driving pressure across CMs was 3.236±1.833mmHg. Red blood cell (RBC) flow was independent of CM resistance, and the ratio of blood flow in adjacent modules was not correlated with their ratio of resistances. In simulated CM geometries, increases to driving pressure produced a direct linear increase to RBC and plasma flow, with no changes to RBC distribution; increases to arteriolar inflow haematocrit resulted in increased RBC flow, but with viscosity-dependent increases to CM resistance. CM RBC flow heterogeneity was higher than plasma flow heterogeneity in experimental data, in contrast to simulated geometries, suggesting that time-dependent flow variability may have important consequences for RBC distribution. In summary, these findings suggest that CMs are active participants in microvascular flow regulation, likely achieved through adjustments to CM driving pressure using pre- and post-capillary loci of flow control. Increases to CM viscosity may be important during the regulation of functional hyperaemia. KEY POINTS: The capillary module (CM), consisting of parallel capillaries from the arteriole to venule, is classically considered to be the building block of capillary networks in skeletal muscle. A detailed evaluation of module haemodynamics may provide insight into mechanisms of blood flow regulation in the microcirculation. Using experimental data from resting skeletal muscle in rats, as well as dual-phase computational models of blood flow, we analysed haemodynamic relationships and the impact of variations to boundary conditions on red blood cell and plasma distribution. We showed that driving pressure across CMs is low, and that simulated increases to inflow haematocrit have important viscosity-dependent effects on module resistance. We found that red blood cell flow was independent from module resistance, which strongly suggests the regulation of driving pressure at the level of the capillary module using pre- and post-capillary loci of flow control. These findings place CMs as central participants in microvascular flow regulation, with important consequences for disease and functional hyperaemia.

Localized Oxygen Exchange Platform for Intravital Video Microscopy Investigations of Microvascular Oxygen Regulation.

Intravital microscopy has proven to be a powerful tool for studying microvascular physiology. In this study, we propose a gas exchange system compatible with intravital microscopy that can be used to impose gas perturbations to small localized regions in skeletal muscles or other tissues that can be imaged using conventional inverted microscopes. We demonstrated the effectiveness of this system by locally manipulating oxygen concentrations in rat extensor digitorum longus muscle and measuring the resulting vascular responses. A computational model of oxygen transport was used to partially validate the localization of oxygen changes in the tissue, and oxygen saturation of red blood cells flowing through capillaries were measured as a surrogate for local tissue oxygenation. Overall, we have demonstrated that this approach can be used to study dynamic and spatial responses to local oxygen challenges to the microenvironment of skeletal muscle.

Two-step machine learning method for the rapid analysis of microvascular flow in intravital video microscopy

Microvascular blood flow is crucial for tissue and organ function and is often severely affected by diseases. Therefore, investigating the microvasculature under different pathological circumstances is essential to understand the role of the microcirculation in health and sickness. Microvascular blood flow is generally investigated with Intravital Video Microscopy (IVM), and the captured images are stored on a computer for later off-line analysis. The analysis of these images is a manual and challenging process, evaluating experiments very time consuming and susceptible to human error. Since more advanced digital cameras are used in IVM, the experimental data volume will also increase significantly. This study presents a new two-step image processing algorithm that uses a trained Convolutional Neural Network (CNN) to functionally analyze IVM microscopic images without the need for manual analysis. While the first step uses a modified vessel segmentation algorithm to extract the location of vessel-like structures, the second step uses a 3D-CNN to assess whether the vessel-like structures have blood flowing in it or not. We demonstrate that our two-step algorithm can efficiently analyze IVM image data with high accuracy (83%). To our knowledge, this is the first application of machine learning for the functional analysis of microvascular blood flow in vivo.

Model‐based analysis of microcirculatory parameters affecting O 2 transport in skeletal muscle subjected to fixed surface PO 2

Microcirculation in skeletal muscle determines both blood flow distribution and O2 transport. The partial pressure of oxygen (PO2) is a prime candidate to assess in tissue, as PO2 variations herald a functionality decline in skeletal muscle. Traditional non-invasive microcirculatory techniques such as Intra-vital Video Microscopy (IVVM) allowing for blood velocity and capillary saturation (SO2) measurements, are unable to directly assess skeletal muscle tissue PO2. Instead, modern microcirculatory O2 analysis relies on biophysical models to recreate tissue PO2 distributions. (Ellis et al, Microcirc 2012, 19:5). Recently, a continuous coupled partial differential equation (PDE) model of two layers of skeletal muscle coupled via diffusion at their boundaries was developed accounting for tissue O2 diffusion, capillary O2 convection, tissue O2 consumption, and capillary-tissue O2 transfer. This model was developed to study an IVVM protocol involving a controlled exposure of skeletal muscle using an O2 exchange chamber. In the protocol studied, hemodynamic parameters in the skeletal muscle region near to the O2 chamber are experimentally shown to exhibit substantial variations. The prevailing theory is capillary perfusion limits the depth of penetration of the chamber O2, and local hemodynamic parameter responses serve to regulate capillary SO2 in skeletal muscle; this would be towards either a fixed target value or homogeneity with neighboring capillary modules. The coupled PDE two-layer model was developed for steady-state PO2 distributions and was solved using traditional math techniques such as operator decoupling and Fourier decomposition, and exhibited the ability to rapidly and accurately calculate PO2 distributions in skeletal muscle which agreed with experimental values. (Afas and Goldman, Vasc Bio 2020). The solution affords for the unique ability not allowed in previous convection-diffusion analytic models of O2 transport, to observe the interaction of both skeletal muscle layers. This is achieved by assuming baseline hemodynamic parameters in the layer far from the O2 chamber and allowing the near layer to take on a range of parameter variations; these variations can be compared to experimental observations. In this study, the behavior of both layers of skeletal muscle adjacent to an O2 chamber will be modelled by the a-priori fixing of parameters in the region far from the O2 chamber and allowing near-layer parameters to be perturbed. Several parameters, including capillary density φ, velocity vB, inlet saturation SIN, hematocrit HT, and tissue consumption μ will have this variation method applied from previous baselines (Afas et al, Math Biosci 2021, in press), and the resultant two-layer O2 profiles will be visualized, as well as the conditions under which the capillary SO2 is homogenized in both layers of skeletal muscle.

Characterization of the Skeletal Muscle Microvascular Blood Flow Response to Tissue CO 2 Perturbations using a Gas Exchange Chamber

Objective To characterize the skeletal muscle microvascular blood flow response to changing tissue carbon dioxide (CO2) concentrations directly imposed using a microfluidic gas exchange chamber. Methods Eight male Sprague-Dawley rats (145-187g) were anaesthetized using sodium pentobarbital via an intraperitoneal injection. Cannulas were introduced into the left common carotid artery and right jugular vein for blood pressure and heart rate monitoring, administration of maintenance anaesthetic, and fluid resuscitation. The animal was then tracheotomized and mechanically ventilated. The extensor digitorum longus muscle of the right hindlimb was isolated, externalized, and reflected over a microfluidic gas exchange chamber set into the stage of an inverted Olympus IX73 microscope for in vivo visualization of blood flow. The muscle was allowed to equilibrate for 30 minutes before blood sampling and exposure to various gas levels. Using computer-controlled mass flow meters, the muscle was equilibrated with incremental levels of CO2 (0-10%) while maintaining a constant O2 state. One-minute intravital video microscopy sequences of the microcirculation were recorded at a range of CO2 levels (0-10%) while maintaining a constant O2 level. Analysis of hemodynamic variables, red blood cell (RBC) velocity, RBC supply rate (SR), RBC oxygen saturation (SO2), and hematocrit, was conducted offline using custom MATLAB software. Results Both the mean RBC velocity and SR increased incrementally as the CO2 increased. The graded increase of both parameters was significant between the low CO2 levels (0-2%) and moderate-high levels (4-10%); however, there was no significant difference between a physiologically relevant CO2 (5%) and elevated CO2 levels (6-10%) (See Figures 1A & 1B). The graded increase in CO2 had no significant impact on capillary SO2 or hematocrit (See Figures 1C & 1D). Conclusion The preliminary findings of the present study demonstrate the local CO2-sensitivity of the skeletal muscle microvascular system using a novel experimental technique. An expected graded increase in RBC velocity and SR were observed as CO2 increased; however, the lack of significant change following exposure to elevated CO2 suggests a higher relative sensitivity of the microvascular system to changes at low tissue CO2 concentrations. The observed invariance of SO2 at varying CO2 gas concentrations also suggests that local CO2-mediated blood flow regulation is not a result of RBC O2 saturation dependent ATP release. Direct effects of CO2 and the resulting change in pH are known to modulate microvascular blood flow, though the underlying mechanisms of CO2-mediated regulation remain to be defined.

The capillary fascicle in skeletal muscle: Structural and functional physiology of RBC distribution in capillary networks.

The capillary module, consisting of parallel capillaries from arteriole to venule, is classically considered as the building block of complex capillary networks. In skeletal muscle, this structure fails to address how blood flow is regulated along the entire length of the synchronously contracting muscle fibres. Using intravital video microscopy of resting extensor digitorum longus muscle in rats, we demonstrated the capillary fascicle as a series of interconnected modules forming continuous columns that align naturally with the dimensions of the muscle fascicle. We observed structural heterogeneity for module topology, and functional heterogeneity in space and time for capillary-red blood cell (RBC) haemodynamics within a module and between modules. We found that module RBC haemodynamics were independent of module resistance, providing direct evidence for microvascular flow regulation at the level of the capillary module. The capillary fascicle is an updated paradigm for characterizing blood flow and RBC distribution in skeletal muscle capillary networks. Capillary networks are the fundamental site of oxygen exchange in the microcirculation. The capillary module (CM), consisting of parallel capillaries from terminal arteriole (TA) to post-capillary venule (PCV), is classically considered as the building block of complex capillary networks. In skeletal muscle, this structure fails to address how blood flow is regulated along the entire length of the synchronously contracting muscle fibres, requiring co-ordination from numerous modules. It has previously been recognized that TAs and PCVs interact with multiple CMs, creating interconnected networks. Using label-free intravital video microscopy of resting extensor digitorum longus muscle in rats, we found that these networks form continuous columns of linked CMs spanning thousands of microns, herein denoted as the capillary fascicle (CF); this structure aligns naturally with the dimensions of the muscle fascicle. We measured capillary-red blood cell (RBC) haemodynamics and module topology (n=9 networks, 327modules, 1491 capillary segments). The average module had length 481μm, width 157μm and 9.51parallel capillaries. We observed structural heterogeneity for CM topology, and functional heterogeneity in space and time for capillary-RBC haemodynamics within a module and between modules. There was no correlation between capillary RBC velocity and lineal density. A passive inverse relationship between module length and haemodynamics was remarkably absent, providing direct evidence for microvascular flow regulation at the level of the CM. In summary, the CF is an updated paradigm for characterizing RBC distribution in skeletal muscle, and strengthens the theory of capillary networks as major contributors to the signal that regulates capillary perfusion.

Detecting cells in intravital video microscopy using a deep convolutional neural network

The analysis of leukocyte recruitment in intravital video microscopy (IVM) is essential to the understanding of inflammatory processes. However, because IVM images often present a large variety of visual characteristics, it is hard for an expert human or even conventional machine learning techniques to detect and count the massive amount of cells and extract statistical measures precisely. Convolutional neural networks are a promising approach to overcome this problem, but due to the difficulty of labeling cells, large data sets with ground truth are rare. The present work explores an adaptation of the RetinaNet model with a suite of augmentation techniques and transfer learning for detecting leukocytes in IVM data. The augmentation techniques include simulating the Airy pattern and motion artifacts present in microscopy imaging, followed by traditional photometric, geometric and smooth elastic transformations to reproduce color and shape changes in cells. In addition, we analyzed the use of different network backbones, feature pyramid levels, and image input scales. We have found that even with limited data, our strategy not only enables training without overfitting but also boosts generalization performance. Among several experiments, the model reached a value of 94.84 for the average precision (AP) metric as our best outcome when using data from different image modalities. We also compared our results with conventional image processing techniques and open-source tools. The results showed an outstanding precision of the method compared with other approaches, presenting low error rates for cell counting and centroid distances. Code is available at: https://github.com/brunoggregorio/retinanet-cell-detection.