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Objectives/hypothesis: To determine if electron paramagnetic resonance (EPR) oximetry is a viable technology to aid in flap monitoring., Study Design: Prospective cohort., Methods: This was a cohort study assessing accuracy and speed of EPR oximetry in detecting ischemia of a saphenous artery-based flap in a rat model, using transcutaneous oximetry as a control. Measurements were obtained under both resting and ischemic conditions for nine Sprague Dawley rats (18 flaps), for 3 postoperative days following flap elevation., Results: The mean partial pressure of oxygen prior to tourniquet application was 66.9 ± 8.9 mm Hg with EPR oximetry and 64.7 ± 5.2 mm Hg with transcutaneous oximetry (P = .45). Mean partial pressures of oxygen during tourniquet application were 8.9 ± 3.2 mm Hg and 8.5 ± 2.9 mm Hg for EPR oximetry and transcutaneous oximetry, respectively (P = .48), and 67.2 ± 6.9 mm Hg and 65.3 ± 6.1 mm Hg after tourniquet release for EPR oximetry and transcutaneous oximetry, respectively (P = .44). The mean ischemia detection time of EPR oximetry was 49 ± 21 seconds., Conclusions: Offering timely, accurate, and noninvasive tissue oxygen measurements, EPR oximetry is a promising adjunct in flap monitoring., Level of Evidence: NA Laryngoscope, 129:E415-E419, 2019., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

We noninvasively monitored the partial pressure of oxygen (pO2) in rat's small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30% of blood flow for experimental rats. Noninvasive measurement of pO2 was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO2 for control rats remained stable over the 7-day period. The pO2 on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO2 post banding with a progressive decline to a final pO2 of 20.9 ± 4.5 mmHg (mean ± SE; p = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe™ was used to validate the low tissue pO2. Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO2 values had a higher intensity of staining. Thus, monitoring SI pO2 using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO2 in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease.

Acute and chronic intestinal ischemia has been linked to the development of gastrointestinal symptoms such as abdominal pain, nausea and vomiting, bowel dysfunction and more seriously, the complications of sepsis, shock and death. Advances in electron paramagnetic resonance (EPR) oximetry have resulted in accurate and reliable in vivo measurement of the partial pressure of oxygen (pO(2)) in solid organs (e.g., muscle, heart) [1], but has yet to be tested in thin walled organs such as intestine. Our ultimate goal is to noninvasively monitor intestinal pO(2) during acute and chronic intestinal ischemia in a rat model. A series of experiments to deliver oxygen-sensitive indicator probes to the small/large intestine by intravenous, luminal and wall injection, as well as direct placement of a solid probe against the outer intestinal wall were attempted. Only the LiNc:BuO:PDMS chip sutured to the peritoneal wall and in direct contact with the intestine allowed for noninvasive pO(2) measurement by EPR. However, the validity of site-specific intestinal pO(2) measurement could not be confirmed and the obtained pO(2) value likely reflected peritoneal cavity oxygenation. Developing methods for probe placement on or inside the intestinal wall are needed for noninvasive, site-specific intestinal pO(2) measurement by EPR to track changes during acute and chronic intestinal ischemia.

Ischemia complicates wound closure. Here, we are unique in presenting a murine ischemic wound model that is based on bipedicle flap approach. Using this model of ischemic wounds we have sought to elucidate how microRNAs may be implicated in limiting wound re-epithelialization under hypoxia, a major component of ischemia. Ischemia, evaluated by laser Doppler as well as hyperspectral imaging, limited blood flow and lowered tissue oxygen saturation. EPR oximetry demonstrated that the ischemic wound tissue had pO(2) <10 mm Hg. Ischemic wounds suffered from compromised macrophage recruitment and delayed wound epithelialization. Specifically, epithelial proliferation, as determined by Ki67 staining, was compromised. In vivo imaging showed massive hypoxia inducible factor-1alpha (HIF-1alpha) stabilization in ischemic wounds, where HIF-1alpha induced miR-210 expression that, in turn, silenced its target E2F3, which was markedly down-regulated in the wound-edge tissue of ischemic wounds. E2F3 was recognized as a key facilitator of cell proliferation. In keratinocytes, knock-down of E2F3 limited cell proliferation. Forced stabilization of HIF-1alpha using Ad-VP16- HIF-1alpha under normoxic conditions up-regulated miR-210 expression, down-regulated E2F3, and limited cell proliferation. Studies using cellular delivery of miR-210 antagomir and mimic demonstrated a key role of miR-210 in limiting keratinocyte proliferation. In summary, these results are unique in presenting evidence demonstrating that the hypoxia component of ischemia may limit wound re-epithelialization by stabilizing HIF-1alpha, which induces miR-210 expression, resulting in the down-regulation of the cell-cycle regulatory protein E2F3.

The nitric oxide (NO)/cGMP pathway, by relaxing vascular smooth muscle cells, is a major physiologic regulator of tissue perfusion. We now identify thrombospondin-1 as a potent antagonist of NO for regulating F-actin assembly and myosin light chain phosphorylation in vascular smooth muscle cells. Thrombospondin-1 prevents NO-mediated relaxation of precontracted vascular smooth muscle cells in a collagen matrix. Functional magnetic resonance imaging demonstrated that an NO-mediated increase in skeletal muscle perfusion was enhanced in thrombospondin-1-null relative to wild-type mice, implicating endogenous thrombospondin-1 as a physiologic antagonist of NO-mediated vasodilation. Using a random myocutaneous flap model for ischemic injury, tissue survival was significantly enhanced in thrombospondin-1-null mice. Improved flap survival correlated with increased recovery of oxygen levels in the ischemic tissue of thrombospondin-1-null mice as measured by electron paramagnetic resonance oximetry. These findings demonstrate an important antagonistic relation between NO/cGMP signaling and thrombospondin-1 in vascular smooth muscle cells to regulate vascular tone and tissue perfusion.

Nitric oxide (NO.) generated from nitric oxide synthase (NOS) isoforms bound to cellular membranes may serve to modulate oxidative stresses in cardiac muscle and thereby regulate the function of key membrane-associated enzymes. Ischemia is known to inhibit the function of sarcolemmal enzymes, including the (Na+ + K+)-ATPase, but it is unknown whether concomitant injury to sarcolemma (SL)-associated NOS isoforms may contribute to this process by reducing the availability of locally generated NO. Here we report that nNOS, as well as eNOS (SL NOSs), are tightly associated with cardiac SL membranes in several different species. In isolated perfused rat hearts, global ischemia caused a time-dependent irreversible injury to cardiac SL NOSs and a disruption of SL NO. generation. Pretreatment with low concentrations of the NO. donor 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) markedly protected both SL NOS and (Na+ + K+)-ATPase functions against ischemia-induced inactivation. Moreover, ischemia impaired SL Na+/K+ binding, and NOC-7 significantly prevented ischemic injury to the ion binding sites on (Na+ + K+)-ATPase. These novel findings indicate that NO. can protect cardiac SL NOSs and (Na+ + K+)-ATPase against ischemia-induced inactivation and suggest that locally generated NO. may serve to regulate SL Na+/K+ ion active transport in the heart.

Stem cells transplanted to the ischemic myocardium usually encounter massive cell death within a few days of therapy. Hypoxic preconditioning (HPC) is currently employed as a strategy to prepare stem cells for increased survival and engraftment in the heart. However, HPC of stem cells has provided varying results, supposedly due to the differ- ences in the oxygen concentration, duration of exposure, and passage conditions. In the present study, we determined the effect of HPC on rat mesenchymal stem cells (MSCs) exposed to 0.5% oxygen concen- tration for 24, 48, or 72 h. We evaluated the expression of prosurvival, proangiogenic, and functional markers such as hypoxia-inducible factor-la, VEGF, phosphorylated Akt, survivin, p21, cytochrome c, caspase-3, caspase-7, CXCR4, and c-Met. MSCs exposed to 24-h hypoxia showed reduced apoptosis on being subjected to severe hypoxic conditions. They also had significantly higher levels of prosurvival, proangiogenic, and prodifferentiation proteins when compared with longer exposure (72 h). Cells taken directly from the cryopreserved state did not respond effectively to the 24-h HPC as those that were cultured under normoxia before HPC. Cells cultured under normoxia before HPC showed decreased apoptosis, enhanced expression of connexin-43, cardiac myosin heavy chain, and CD31. The preconditioned cells were able to differentiate into the cardiovas- cular lineage. The results suggest that MSCs cultured under normoxia before 24-h HPC are in a state of optimal expression of prosurvival, proangiogenic, and functional proteins that may increase the survival and engraftment in the infarct heart. These results could provide further insights into optimal preparation of MSCs which would greatly influence the effectiveness of cell therapy in vivo. [ABSTRACT FROM AUTHOR]

Acute ischemic stroke (AIS) results in focal deprivation of blood-borne factors, one of them being oxygen. The purpose of this study was two-fold: (1) to identify therapeutic conditions for supplemental oxygen in AIS and (2) to use transcriptome-wide screening toward uncovering oxygen-sensitive mechanisms. Transient MCAO in rodents was used to delineate the therapeutic potential of normobaric (NBO, 100% O2, 1ATA) and hyperbaric oxygen (HBO, 100% O2, 2ATA) during ischemia (iNBO, iHBO) and after reperfusion (rNBO, rHBO). Stroke lesion was quantified using 4.7 T MRI at 48 h. Supplemental oxygen during AIS significantly attenuated percent stroke hemisphere lesion volume as compared with that in room air (RA) controls, whereas identical treatment immediately after reperfusion exacerbated lesion volume (RA=22.4±1.8, iNBO=9.9±3.6, iHBO=6.6±4.8, rNBO=29.8±3.6, rHBO=35.4±7.6). iNBO and iHBO corrected penumbra tissue pO2 during AIS as measured by EPR oxymetry. Unbiased query of oxygen-sensitive transcriptome in stroke-affected tissue identified 5,769 differentially expressed genes. Candidate genes were verified by real-time PCR using neurons laser-captured from the stroke-affected somatosensory cortex. Directed microarray analysis showed that supplemental oxygen limited leukocyte accumulation to the infarct site by attenuation of stroke-inducible proinflammatory chemokine response. The findings provide key information relevant to understanding oxygen-dependent molecular mechanisms in the AIS-affected brain. [ABSTRACT FROM AUTHOR]

Background: Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP). Methods and Findings: Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups. Conclusion: Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction. [ABSTRACT FROM AUTHOR]

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation (r² = 0.85) between measured MRI and triphenyltetrazolium chloride (TIC) infarct volumes was noted. Our observation that until day 3 post-I/R the infarct size as measured by TIC staining and MRI was much larger than that of the myocyte-silent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MRI and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time. [ABSTRACT FROM AUTHOR]

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 mm of global ischemia at 37°C followed by 45 mm of reperfusion. Hearts were perfused with PC (10 μ.M) or Spirulina preparation (SP, 50 mg/I) for 15 mm before the onset of ischemia and throughout reperfusion. After 45 mm of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK 1⁄2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1⁄2. [ABSTRACT FROM AUTHOR]

Postischemic myocardial contractile dysfunction is in part mediated by the burst of reactive oxygen species (ROS), which occurs with the reintroduction of oxygen. We hypothesized that tissue oxygen tension modulates this ROS burst at reperfusion. After 20 mm of global ischemia, isolated rat hearts were reperfused with temperature-controlled (37.4°C) Krebs-Henseleit buffer saturated with one of three different O2 concentrations (95, 20, or 2%) for the first 5 mm of reperfusion and then changed to 95% O2. Additional hearts were loaded with I) allopurinol (1 mM), a xanthine oxidase inhibitor, 2) diphenyleneiodonium (DPI; 1µM), an NAD(P)H oxidase inhibitor, or 3) Tiron (10 mM), a superoxide scavenger, and were then reperfused with either 95 or 2% O2 for the first 5 mi ROS production and tissue oxygen tension were quantitated using electron paramagnetic resonance spectroscopy. Tissue oxygen tension was significantly higher in the 95% O2 group. However, the largest radical burst occurred in the 2% O2 reperfusion group (P < 0.001). Recovery of left ventricular (LV) contractile function and aconitase activity during reperfusion were inversely related to the burst of radical production and were significantly higher in hearts initially reperfused with 95% O2 (P < 0.001). Allopurinol, DPI, and Tiron reduced the burst of radical formation in the 2% O2 reperfusion groups (P < 0.05). Hypoxic reperfusion generates an increased ROS burst originating from multiple pathways. Recovery of LV function during reperfusion is inversely related to this oxygen radical burst, highlighting the importance of myocardial oxygen tension during initial reperfusion. [ABSTRACT FROM AUTHOR]

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42°C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration. [ABSTRACT FROM AUTHOR]

Presents information on a study which demonstrated that active neutrophils are a major source of oxidants in hearts reperfused in vivo after prolonged ischemia. Blood sampling for oxygen radical measurements; Electron paramagnetic resonance spectroscopy; Effect of antineutrophil antibodies.

Summary: Aim of study: Previous reports note an increase in both reactive oxygen species (ROS) and nitric oxide (e onset of myocardial reperfusion. We tested the hypothesis that inhibition of production at the time of reperfusion improves recovery of post-ischemic myocardial function. Methods and materials: Isolated rat hearts were perfused with temperature controlled (37.4°C) modified Krebs Henseleit buffer solution at 85mm Hg. Following 20min of global ischemia, hearts were reperfused for the first 10min with: (1) standard buffer (control), (2) buffer with a NOS inhibitor, N-nitro-l-arginine methyl ester (l-NAME), (3) buffer with superoxide dismutase (SOD) or (4) buffer with N-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite generator. Tissue O2 and ontinuously measured with thin electrochemical probes embedded in the wall of the LV. ROS was measured with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (40mM). LV contractile function was continuously monitored. Results: Recovery of LV contractile function was significantly improved in hearts initially reperfused with l-NAME and SOD and significantly depressed in hearts reperfused with SIN-1 compared with control (p <0.01, n =5–8 per group). DMPO-adduct during reperfusion (measure of ROS) was significantly decreased with SOD (p <0.001 versus l-NAME and Control, n =4 per group) and unchanged with l-NAME and SIN-1 compared with Control. With l-NAME, tissue 2 were significantly decreased, independent of coronary flow, during reperfusion compared with control and SIN-1. Conclusions: Inhibition of O2 − or time of reperfusion improves early reperfusion LV function and alters tissue oxygen tension. In contrast to pre-ischemic treatments, intervention to reduce peroxynitrite generation at the onset of reperfusion can effectively improve post-ischemic myocardial recovery. [Copyright &y& Elsevier]

Abstract: Trimetazidine, the known anti-anginal and anti-ischemic drug, was modified by pyrroline and tetrahydropyridine nitroxides and their hydroxylamine and sterically hindered secondary amine precursors. The synthesized new compounds proved to be better superoxide scavenger molecules compared to the parent Trimetazidine in an in vitro experiment. This reactive oxygen species (ROS) scavenging activity was further supported by ischemia/reperfusion (I/R) studies on Langendorff-perfused rat hearts pretreated with Trimetazidine and with the modified Trimetazidine derivatives before ischemia. Two of the investigated compounds, containing 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole and 4-phenyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole substituents on the piperazine ring, provided significant protection from the cardiac dysfunction caused by I/R. The protective effect could be attributed to the combined anti-ischemic and antioxidant effects. [Copyright &y& Elsevier]