Microvasculature in murine tracheal allografts after combined therapy with clopidogrel and everolimus

OBJECTIVES Survival after lung transplantation is mainly limited by the development of chronic lung allograft dysfunction. Previous studies have suggested T-cell mediated proliferation and microvascular changes in experimental small airways models as potential therapeutic targets. The aim of this study was to assess microvascular changes in murine orthotopic tracheal allografts after treatment with everolimus alone or in combination with clopidogrel. METHODS C57Bl/6 (H-2b) donor tracheas were orthotopically transplanted into CBA (H-2k) recipients. Mice received daily injections of everolimus (0.05 mg/kg) alone or combined with clopidogrel (1 mg/kg). Twenty-eight days after transplantation, ratio of the thickness of tracheal epithelium and lamina propria was measured as an indicator for chronic rejection. Additionally, graft oxygenation and graft perfusion were detected on postoperative days 4, 10 and 28. Quantitative reverse transcription polymerase chain reaction analysis was used for gene expression analysis. RESULTS While syngeneic grafts showed a stable tissue pO2 and undisturbed microvascular perfusion, rejecting allografts had a drastic decline in both parameters as well as a flattened epithelium and an increased thickness of the lamina propria. Treatment with everolimus reduced allogeneic fibroproliferation, but had no protective effects on the microvasculature; polymerase chain reaction analysis indicated hypoxic stress and inflammation. Combining everolimus with clopidogrel improved microvascular integrity in the tracheal grafts, but had no synergistic effect in preventing obliterative bronchiolitis development. CONCLUSIONS These data demonstrate that the ability of everolimus to reduce the development of post-transplant obliterative bronchiolitis is not caused by microvascular protection and has no synergistic effects with clopidogrel in acute airway rejection.

Renal tissue Po2 sensing during acute hemodilution is dependent on the diluent

Sensing changes in blood oxygen content ([Formula: see text]) is an important physiological role of the kidney; however, the mechanism(s) by which the kidneys sense and respond to changes in [Formula: see text] are incompletely understood. Accurate measurements of kidney tissue oxygen tension ([Formula: see text]) may increase our understanding of renal oxygen-sensing mechanisms and could inform decisions regarding the optimal fluid for intravascular volume resuscitation to maintain renal perfusion. In some clinical settings, starch solution may be nephrotoxic, possibly due to inadequacy of tissue oxygen delivery. We hypothesized that hemodilution with starch colloid solutions would reduce [Formula: see text] to a more severe degree than other diluents. Anesthetized Sprague-Dawley rats ( n = 77) were randomized to undergo hemodilution with either colloid (6% hydroxyethyl starch or 5% albumin), crystalloid (0.9% saline), or a sham procedure (control) ( n = 13–18 rats/group). Data were analyzed by ANOVA with significance assigned at P < 0.05. After hemodilution, mean arterial pressure (MAP) decreased marginally in all groups, while hemoglobin (Hb) and [Formula: see text] decreased in proportion to the degree of hemodilution. Cardiac output was maintained in all groups after hemodilution. [Formula: see text] decreased in proportion to the reduction in Hb in all treatment groups. At comparably reduced Hb, and maintained arterial oxygen values, hemodilution with starch resulted in larger decreases in [Formula: see text] relative to animals hemodiluted with albumin or saline ( P < 0.008). Renal medullary erythropoietin (EPO) mRNA levels increased more prominently, relative to other hypoxia-regulated molecules (GLUT-1, GAPDH, and VEGF). Our data demonstrate that the kidney acts as a biosensor of reduced [Formula: see text] following hemodilution and that [Formula: see text] may provide a quantitative signal for renal cellular responsiveness to acute anemia. Evidence of a more severe reduction in [Formula: see text] following hemodilution with starch colloid solution suggests that tissue hypoxia may contribute to starch induced renal toxicity.

Electrochemical carbon fiber-based technique for simultaneous recordings of brain tissue PO2, pH, and extracellular field potentials

A method for simultaneous electrochemical detection of brain tissue PO2 (PtO2) and pH changes together with neuronal activity using a modified form of fast cyclic voltammetry with carbon fiber electrodes is described. This technique has been developed for in vivo applications and recordings from discrete brain nuclei in experimental animals. The small size of the carbon fiber electrode (⍰7μm, length <100μm) ensures minimal disruption of the brain tissue and allows recordings from small brain areas. Sample rate (up to 4 Hz) is sufficient to resolve rapid changes in PtO2 and pH that follow changes in neuronal activity and metabolism. Rapid switching between current and voltage recordings allows combined electrochemical detection and monitoring of extracellular action potentials. For simultaneous electrochemical detection of PtO2 and pH, two consecutive trapezoidal voltage ramps are applied with double differential-subtraction of the background current. This enables changes in current caused by protons and oxygen to be detected separately with minimal interference between the two. The profile of PtO2 changes evoked by increases in local neuronal activity recorded using the described technique was similar to that of blood oxygen level dependent responses recorded using fMRI. This voltammetric technique can be combined with fMRI and brain vessel imaging to study the metabolic mechanisms underlying neurovascular coupling response with much greater spatial and temporal resolution than is currently possible.

A Computer Model of Oxygen Dynamics in the Cortex of the Rat Kidney at the Cell-Tissue Level

The renal cortex drives renal function. Hypoxia/reoxygenation are primary factors in ischemia-reperfusion (IR) injuries, but renal oxygenation per se is complex and awaits full elucidation. Few mathematical models address this issue: none captures cortical tissue heterogeneity. Using agent-based modeling, we develop the first model of cortical oxygenation at the cell-tissue level (RCM), based on first principles and careful bibliographical analysis. Entirely parameterized with Rat data, RCM is a morphometrically equivalent 2D-slice of cortical tissue, featuring peritubular capillaries (PTC), tubules and interstitium. It implements hemoglobin/O2 binding-release, oxygen diffusion, and consumption, as well as capillary and tubular flows. Inputs are renal blood flow RBF and PO2 feeds; output is average tissue PO2 (tPO2). After verification and sensitivity analysis, RCM was validated at steady-state (tPO2 37.7 ± 2.2 vs. 36.9 ± 6 mmHg) and under transients (ischemic oxygen half-time: 4.5 ± 2.5 vs. 2.3 ± 0.5 s in situ). Simulations confirm that PO2 is largely independent of RBF, except at low values. They suggest that, at least in the proximal tubule, the luminal flow dominantly contributes to oxygen delivery, while the contribution of capillaries increases under partial ischemia. Before addressing IR-induced injuries, upcoming developments include ATP production, adaptation to minutes–hours scale, and segmental and regional specification.

Pharmacological Increase of Hb-O2 Affinity with a Voxelotor Analog Does Not Decrease Brain Tissue pO2 or Limit O2 Extraction in Brain Tissues of Sickle Cell Mice

Sickle Cell Disease (SCD) is characterized by hemolytic anemia, vaso-occlusion, and progressive end-organ damage. The underlying mechanism of SCD is the polymerization of sickle hemoglobin (HbS) that occurs when sickle erythrocytes (SS RBCs) are partially deoxygenated in microcirculation, leading to SCD pathophysiologic features. One of the most devastating complications of SCD occurs in the central nervous system (CNS), where overt stroke or repeated silent cerebral infarcts lead to significant physical and neurocognitive consequences. In SCD, the brain's response to insufficient oxygen (O2) delivery is balanced by increased blood flow to preserve O2 supply. However, the systemic endothelial dysfunction in SCD limits the capacity for vascular regulatory and compensatory changes to preserve appropriate tissue oxygenation, especially in tissues with high O2 demand like the brain. Low hemoglobin (Hb) levels and increased cerebral blood flow (CBF) are associated with increased stroke risk, suggesting that anemia-induced tissue hypoxia is an important factor contributing to subsequent morbidity in SCD patients. Voxelotor (GBT440) is a small molecule, HbS polymerization inhibitor being developed by Global Blood Therapeutics (GBT) for the treatment of SCD. By addressing the underlying mechanism of SCD, voxelotor has the potential to be disease-modifying and alleviate the clinical manifestations of SCD. Mechanistically, voxelotor increases Hb-O2 affinity and delays the transition from oxyHb to deoxyHb under hypoxic conditions. This study assessed the impact of a pharmacologically mediated increase in Hb-O2 affinity on brain tissue oxygenation under both normoxic and hypoxic conditions in Townes transgenic sickle mice (SCD mice). Two compounds that increase the Hb-O2 affinity with similar potency, voxelotor and an analog to voxelotor, GBT1118, were considered for the study. The target for Hb occupancy with test compounds was ≥30% based on the therapeutic target occupancies observed with voxelotor in clinical studies. The effects of increased Hb-O2 affinity on brain tissue oxygenation were assessed directly with O2-specific microelectrodes in a cranial window and indirectly with hypoxyprobe staining (pimonidazole) of brain tissue. Unique to this SCD model, the targeted Hb occupancy (≥30%) could not be consistently achieved by voxelotor. In contrast, repeat oral dosing of GBT1118 at 200 mg/kg/day for 2 weeks in SCD mice achieved steady state concentrations of 802 ± 81 µM (mean ± SD; n=5), corresponding to a Hb occupancy of 44 ± 5%. Consequently, GBT1118 decreased the p50 (partial pressure of O2 at which Hb is 50% saturated) values of SCD mouse blood from 39 ± 0.8 mmHg (Vehicle-dosed) to 21 ± 1.6 mmHg (GBT1118-dosed). While we could not achieve the desired Hb occupancy (≥30%) with voxelotor in this model, the Hb occupancy and change in p50 achieved with GBT1118 afforded us the opportunity to ask whether significantly increasing Hb-O2 affinity affects brain O2 tension. Measurements of cortical O2 tension (pO2) showed no difference in pO2 values under normoxia (21% O2) (Figure A), and slightly higher pO2 values under hypoxia (10% O2) (Figure B) for GBT1118-dosed SCD mice compared with vehicle-dosed SCD mice. Collectively across all brain tissues, the GBT1118-induced increase in Hb-O2 affinity reduced tissue hypoxia in SCD mice under hypoxia as measured by pimonidazole staining (Figure C). Together, these results indicate that a pharmacological increase of Hb-O2 affinity does not decrease cortical tissue pO2 in SCD mice and may reduce brain hypoxia under hypoxic conditions. Figure Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Lucas:Global Blood Therapeutics: Research Funding. Muller:Global Blood Therapeutics: Research Funding. Williams:Global Blood Therapeutics: Research Funding. Zhang:Global Blood Therapeutics: Employment, Equity Ownership. Rademacher:Global Blood Therapeutics: Employment, Equity Ownership. Alt:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Research Funding.

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2–3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation.