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Human populations are experiencing unprecedented growth and longevity with lingering knowledge gaps of the characteristics, mechanisms, and pathologies of senescence. Invasive measurements and long-term control conditions for longitudinal studies are infeasible, necessitating the need for surrogate animal models. Rats have short lifespans (2-3 years) with translatable cardiovascular systems, and Sprague Dawley microcirculatory preparations are key to studying the oxygen transport mechanisms critical to the loss of skeletal muscle function in aging. Here we present baseline physiological data of 61 male, Sprague Dawley rats at 3, 6, 12, 18, and 24 months of age. Anesthetized animals were surgically prepared for femoral arterial and venous cannulations, tracheal intubation, and exteriorization of the spinotrapezius muscle. Measurements included cardiovascular function, blood gases, and peripheral tissue interstitial oxygen tension (P ISF O 2 ) using phosphorescence quenching microscopy. Intrinsic heart rates decreased with age without significant changes to blood pressure. Arterial oxygen tension declined 17% by 18 and 24 Months (p < 0.05) while p A CO 2 and P ISF O 2 were unchanged. Lactate was elevated at 12 and 18 Months along with an alkaline shift in blood pH. Heart rate and decreased p A O 2 decoupled from p A CO 2 are conserved phenomena in human aging. The continuity of resting P ISF O 2 despite an anaerobic shift in metabolism may be due to declining mitochondrial function and dysregulation of the vascular response to hypoxemia, which are also present in aged humans. These physiological and microcirculatory data offer a useful experimental model for investigating the detailed changes in oxygen supply and demand that affect senescing skeletal muscles in rats and humans., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Acute respiratory distress syndrome (ARDS) is a complication of pulmonary disease that produces life-threatening hypoxaemia. Despite ventilation and hyperoxic therapies, undetected hypoxia can manifest in capillary beds leading to multi-organ failure. Ox66™ is an ingestible, solid-state form of oxygen designed to supplement oxygen deficits. Twenty-four anaesthetized rats underwent a two-hit model of respiratory distress (ARDS), where a single dose (5 mg/kg) of lipopolysaccharide (LPS) was given intratracheally, and then the respiratory tidal volume was reduced by 40%. After 60 min, animals were randomized to receive Ox66™, or normal saline (NS; vehicle control) via gavage or supplemental inspired oxygen (40% FiO 2 ). A second gavage was administered at 120 min. Cardiovascular function and blood oximetry/chemistry were measured alongside the peripheral spinotrapezius muscle's interstitial oxygenation (P ISF O 2 ). ARDS reduced mean arterial pressure (MAP) and P ISF O 2 compared to baseline (BL) for all treatment groups. Treatment with Ox66 or NS did not improve MAP, but 40% FiO 2 caused a rapid return to BL. P ISF O 2 improved after treatment with Ox66 ™ and 40% FiO 2 and remained elevated for both groups against NS until study conclusion. Both oxygen treatments also suppressed the inflammatory response to LPS, suggesting that Ox66 ™ can deliver therapeutically-impactful levels of oxygen in situations of pulmonary dysfunction.

Abstract: Blood products are the current standard for resuscitation of hemorrhagic shock. However, logistical constraints of perishable blood limit availability and prehospital use, meaning alternatives that provide blood-like responses remain an area of active investigation and development. VS-101 is a new PEGylated human hemoglobin-based oxygen carrier that avoids the logistical hurdles of traditional blood transfusion. This study sought to determine the safety and ability of VS -101 to maintain circulatory function and capillary oxygen delivery in a severe (50%) exchange transfusion (ET) model. Anesthetized, male Sprague Dawley rats were prepared for cardiovascular monitoring and phosphorescence quenching microscopy of interstitial fluid oxygen tension (P ISFo2 ) in the spinotrapezius muscle. Fifty-percent isovolemic ET of estimated total blood volume with either lactated Ringer's solution (LRS, n = 8) or VS -101 (n = 8) at 1 mL/kg/min was performed, and animals were observed for 240 min. VS -101 maintained P ISFo2 at baseline with a transient 18 ± 4 mm Hg decrease ( P < 0.05) in mean arterial pressure (MAP). In contrast, ET with LRS decreased P ISFo2 by approximately 50% ( P < 0.05) and MAP by 74 ± 10 mm Hg ( P < 0.05). All VS -101 animals survived 240 min, the experimental endpoint, while 100% of LRS animals expired by 142 min. VS -101 animals maintained normal tissue oxygenation through 210 min, decreasing by 25% ( P < 0.05 vs. baseline) thereafter, likely from VS -101 vascular clearance. No arteriolar vasoconstriction was observed following VS -101 treatment. In this model of severe ET, VS -101 effectively maintained blood pressure, perfusion, and P ISFo2 with no vasoconstrictive effects. Further elucidation of these beneficial resuscitation effects of VS -101 is warranted to support future clinical trials., Competing Interests: The study was funded by Vivosang, Inc., manufacturer of VS-101 and patent holder of another PEG-Hb product, MP4. AM, KV, and WMS are employed by Vivosang, Inc. Manuscript preparation was funded and executed by Song Biotechnologies, LLC. WHN and BKS are employees of Song Biotechnologies. FRS reports no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Sepsis is a devastating complication of infection and injury that, through widespread endothelial dysfunction, can cause perfusion deficits and multi-organ failure. To address the recognised need for therapeutics targetting the endothelial barrier, a topical formulation (CUR; VASCEPTOR™; Vascarta Inc, Summit, NJ) was developed to transdermally deliver bio-active concentrations of curcumin-an anti-inflammatory and nitric oxide promoter. Male, Sprague Dawley rats were treated daily with lipopolysaccharide (LPS, 10 mg/kg, IP) to induce endotoxemia, and topical applications of Vehicle Control (LPS + VC; N  = 7) or Curcumin (LPS + CUR; N  = 7). A third group received neither LPS nor treatment (No-LPS; N  = 8). After 72 h, animals were surgically prepared for measurements of physiology and endothelial dysfunction in the exteriorised spinotrapezius muscle through the extravasation of 67 kDa TRITC-BSA (albumin) and 500 kDa FITC-dextran (dextran). At 72 h, LPS + VC saw weight loss, and increases to pulse pressure, lactate, pCO 2 , CXCL5 (vs No-LPS) and IL-6 (vs 0 h; p  < 0.05). LPS + CUR was similar to No-LPS, but with hypotension. Phenylephrine response was increased in LPS + CUR. Regarding endothelial function, LPS + CUR albumin and dextran extravasation were significantly reduced versus LPS + VC suggesting that Curcumin mitigated endotoxemic endothelial dysfunction. The speculated mechanisms are nitric oxide modulation of the endothelium and/or an indirect anti-inflammatory effect.

This study examined changes in interstitial PO 2 , which allowed calculation of VO 2 during periods of rest, muscle contraction and recovery using an in situ rat spinotrapezius muscle preparation. The PO 2 was measured using phosphorescence quenching microscopy and the muscle VO 2 was calculated as the rate of O 2 disappearance during brief periods of muscle compression to stop blood flow with a supra-systolic pressure. The PO 2 and VO 2 measurements were made during "5 s compression and 15 s recovery" (CR) cycles. With all three stimulation frequencies, 1, 2 and 4 Hz, the fall in interstitial PO 2 and rise in VO 2 from resting values occurred within the first 20 s of contraction. The PO 2 during contraction became lower as stimulation frequency increased from 1 to 4 Hz. VO 2 was higher at 2 Hz than at 1 Hz contraction. With cessation of stimulation, PO 2 began increasing exponentially towards baseline values. After 1 and 2 Hz contraction, the fall in muscle VO 2 was delayed by one CR cycle and then exponentially decreased towards resting values. After 4 Hz stimulation, VO 2 increased for 2 cycles and then decreased. The post-contraction transients of PO 2 and VO 2 were not synchronous and had different time constants. With further analysis two distinct functional responses were identified across all stimulation frequencies having PO 2 during contraction above or below 30 mmHg. The corresponding VO 2 responses were different - for "high" PO 2 , muscle VO 2 reached high levels, while for the "low" PO 2 data set muscle VO 2 remained low. Recovery patterns were similar to those described above. In summary, local microscopic PO 2 and VO 2 were measured in resting and contracting muscle in situ and the post-contraction transients of PO 2 and VO 2 were all much slower than the onset transients., Competing Interests: Authors AG, BS, and WN were employed by Song Biotechnologies LLC. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Golub, Song, Nugent and Pittman.)

Introduction: Pulmonary dysfunction (PD) and its associated hypoxia present a complication to the care of many service members and can arise intrinsically via comorbidities or extrinsically by infection or combat-related trauma (burn, smoke inhalation, and traumatic acute lung injury). Current supportive treatments (e.g., ventilation and supplemental oxygen) relieve hypoxia but carry a significant risk of further lung injury that drives mortality. Ox66 is a novel, solid-state oxygenating compound capable of delivering oxygen via intravenous infusion., Materials and Methods: Male Sprague Dawley rats (N = 21; 250-300 g) were surgically prepared for cardiovascular monitoring, fluid infusion, mechanical ventilation, and intravital and phosphorescence quenching microscopy (interstitial oxygen tension; PISFO2) of the spinotrapezius muscle. Baselines (BL) were collected under anesthesia and spontaneous respiration. PD was simulated via hypoventilation (50% tidal volume reduction) and was maintained for 3 hours. Groups were randomized to receive Ox66, normal saline (NS; vehicle control), or Sham (no treatment) and were treated immediately following PD onset. Arterial blood samples (65 µL) and intravital images were taken hourly to assess blood gases and chemistry and changes in arteriolar diameter, respectively. Significance was taken at P < .05., Results: PD reduced PISFO2 for all groups; however, by 75 minutes, both NS and Sham were significantly lower than Ox66 and remained so until the end of PD. Serum lactate levels were lowest in the Ox66 group-even decreasing relative to BL-but only significant versus Sham. Furthermore, all Ox66 animals survived the full PD challenge, while one NS and two Sham animals died. No significant vasoconstrictive or vasodilative effect was noted within or between experimental groups., Conclusion: Treatment with intravenous Ox66 improved interstitial oxygenation in the spinotrapezius muscle-a recognized bellwether for systemic capillary function-suggesting an improvement in oxygen delivery. Ox66 offers a novel approach to supplemental oxygenation that bypasses lung injury and dysfunction., (© The Association of Military Surgeons of the United States 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

DNA methylation is an epigenetic mechanism controlling gene expression, and reduced methylation is associated with increased gene expression. We hypothesized that IL-17 cytokines are regulated by DNA methylation, are elevated in the circulation of preeclamptic women, and stimulate vascular neutrophil chemokine expression, which could account for vascular infiltration of neutrophils in preeclampsia. We found significantly reduced DNA methylation of IL17A, IL17E, and IL17F genes in omental arteries of preeclamptic women, significantly reduced methylation of IL2, which regulates IL-17-producing T-lymphocytes, and significantly reduced methylation of genes encoding neutrophil chemokines and TNFα receptors related to lymphocyte function. Maternal plasma levels of IL-17A were significantly elevated in the second trimester of preeclamptic pregnancy as compared to normal pregnancy. To test if methylation regulates IL-17 cytokines, a lymphocyte cell line (Jurkat) was cultured with a hypomethylating agent. Hypomethylation increased expression of IL17E (aka IL25), IL17F, and IL2. IL17A was not expressed by Jurkat cells. To test the potential role of IL-17 cytokines in vascular neutrophil infiltration associated with preeclampsia, human vascular smooth muscle cells were cultured with IL-17 cytokines. IL-17A, but not IL-17E or IL-17F, increased gene expression of neutrophil chemokines (IL-8, CXCL5, and CXCL6) that are increased in vascular smooth muscle of preeclamptic women. The monocyte chemokine, CCL-2, was not increased. TNFα also increased neutrophil chemokines. IL-17 cytokines are regulated by DNA methylation; IL-17A is elevated in preeclampsia and stimulates expression of neutrophil chemokines in vascular smooth muscle. IL-17A could be responsible for vascular infiltration of neutrophils in preeclampsia., (© 2021. Society for Reproductive Investigation.)

Acute respiratory distress syndrome (ARDS) features pulmonary dysfunction capable of causing life-threatening hypoxaemia. Ventilation and hyperoxic therapies force oxygen through dysfunctional alveoli but risk exacerbating damage. Ox66™ is an ingestible, solid-state oxygen product designed for oxygen supplementation. Eighteen anaesthetized, ventilated rats were subjected to a 40% reduction in tidal volume to produce a hypoventilatory simulation of the hypoxia in ARDS (HV-ARDS). After 60 min, animals were randomized to receive either normal saline (Saline; volume control) or Ox66 ™ gavage. Cardiovascular function and blood oximetry/chemistry were measured alongside interstitial oxygenation (P ISF O 2 ) of the peripheral spinotrapezius muscle. HV-ARDS reduced mean arterial pressure by ∼20% and P ISF O 2 by ∼35% for both groups. Ox66 ™ gavage treatment at 60 min improved P ISF O 2 over Saline ( p  < .0001), restoring baseline values, however, the effect was temporary. A second bolus at 120 min repeated the OX66 ™ P ISF O 2 response, which remained elevated over Saline ( p  < .01) until study end and was supported by systemic parameters of lactate, P a O 2 , SO 2 , and base deficit. Saline remained hypotensive, whereas Ox66 ™ became normotensive. Vasoconstriction was observed in the Saline, but not Ox66 ™ group. Supplemental oxygenation through Ox66 ™ gavage increased peripheral tissue oxygenation, warranting further study for disorders featuring dysfunction of pulmonary perfusion like ARDS.

Oxygen (O 2 ) exchange between capillaries and muscle cells in exercising muscles is of great interest for physiology and kinesiology. However, methodical limitations prevent O 2 measurements on the millisecond scale. To bypass the constraints of quasi-continuous recording, progressive measurements of O 2 partial pressure (PO 2 ) in rhythmically contracting skeletal muscle were compiled to describe the O 2 kinetics surrounding and including a single muscle contraction. Phosphorescence quenching microscopy measured PO 2 in the interstitium of the rat spinotrapezius muscle. Measurements were triggered by contraction-inducing electrical pulses. For the first 60 seconds, measurement preceeded stimulation. After 60, measurement followed with a progressive 20 ms increment. Thus, the first 60 measurements describe the overall PO 2 response to electrical stimulation initiated after a 10 second rest period, while 61-100 (stroboscopic mode) were compiled into a single 800 ms profile of the PO 2 transient surrounding muscle contraction. Thirty seconds of stimulated contractions decreased interstitial PO 2 from a baseline of 71 ± 1.4 mmHg to an "active" steady-state of 43 ± 1.5 mmHg. The stroboscopic mode compilation revealed an unexpected post-contractile rise in PO 2 as a 205 ms spike with a maximum amplitude of 58 ± 3.8 mmHg at 68 ms, which restored 58% of the PO 2 drop from baseline. Interpretation of this phenomenon is based on classical experiments by G.V. Anrep (1935), who discovered the rapid thrust of blood flow associated with muscle contraction. In addition to the metabolic implications during exercise, the physiological impact of these PO 2 spikes may grow with an increased rate of rhythmical contractions in muscle or heart. NEW&NOTEWORTHY: The principal finding is a spike of interstitial PO 2 , produced by a twitch in a rhythmically contracting muscle. A possible mechanism is flushing capillaries with arterial blood by mechanical forces. A technical novelty is the PO 2 measurement with a "stroboscopic mode" and progressively increasing delay between stimulator pulse and PO 2 measuring. That permitted a 20 ms time resolution for a 205 ms spike duration, using an excitation flash rate one per second., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Medical support for traumatic haemorrhage is lacking for far-forward combat units. VIR-HBOC (haemoglobin-based oxygen carrier) is a novel biological therapeutic under development as a field-stable resuscitation fluid. HBOCs have a long history of complications, chief among them is vasoconstrictive hypertension, which must be resolved before efficacy testing. As such, VIR-HBOC was compared against Lactated Ringers (LRS; vehicle) and a cross-linked haemoglobin (ααHb; a known vasoactive HBOC) in a rat topload model. Twenty-three male, Sprague Dawley rats were randomly assigned to receive a 10% infusion (estimated total blood volume) of one test article while normotensive and under anaesthesia. Cardiovascular, blood chemistry and oximetry, microvascular arteriolar diameters, and interstitial tissue oxygenation parameters were measured. Circulatory half-life was calculated by plasma total haemoglobin. Treatment with ααHb caused immediate increases in mean arterial pressure compared to LRS and VIR-HBOC groups, and corresponding arteriolar vasoconstriction ( p  < .05), which did not occur for LRS or VIR-HBOC. Circulatory half-lives for VIR-HBOC and ααHb were calculated as 340 and 157 min, respectively. This first report of VIR-HBOC showed no evidence of a hypertensive or vasoactive effect. It was well-tolerated over the eight-hour time course of this topload model, which warrants further investigation in studies of haemorrhagic shock.

We tested a novel hypothesis that elevated levels of proteases in the maternal circulation of preeclamptic women activate neutrophils due to their pregnancy-specific expression of protease-activated receptor 1 (PAR-1). Plasma was collected longitudinally from normal pregnant and preeclamptic women and analyzed for MMP-1 and neutrophil elastase. Neutrophils were isolated for culture and confocal microscopy. Omental fat was collected for immunohistochemistry. Circulating proteases were significantly elevated in preeclampsia. Confocal microscopy revealed that tet methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, and p65 subunit of NF-κB were strongly localized to the nucleus of untreated neutrophils of preeclamptic women, but in untreated neutrophils of normal pregnant women they were restricted to the cytosol. Treatment of normal pregnancy neutrophils with proteases activated PAR-1, leading to activation of RhoA kinase (ROCK), which triggered translocation of TET2 and p65 from the cytosol into the nucleus, mimicking the nuclear localization in neutrophils of preeclamptic women. IL-8, an NF-κB-regulated gene, increased in association with TET2 and p65 nuclear localization. Co-treatment with inhibitors of PAR-1 or ROCK prevented nuclear translocation and IL-8 did not increase. Treatment of preeclamptic pregnancy neutrophils with inhibitors emptied the nucleus of TET2 and p65, mimicking the cytosolic localization of normal pregnancy neutrophils. Expression of PAR-1 and TET2 were markedly increased in omental fat vessels and neutrophils of preeclamptic women. We conclude that elevated levels of circulating proteases in preeclamptic women activate neutrophils due to their pregnancy-specific expression of PAR-1 and speculate that TET2 DNA de-methylation plays a role in the inflammatory response.

Introduction: Hemorrhage is a leading cause of death from potentially survivable civilian and military trauma. As projected conflicts move from settings of tactical and logistical supremacy to hyper-dynamic tactical zones against peer and near-peer adversaries, protracted medical evacuation times are expected. Treatment at the point-of-injury is critical. Although crystalloids like Lactated Ringer's (LR) are ubiquitous, whole blood (WB) is the preferred resuscitation fluid following hemorrhage; however, logistical constraints limit the availability of WB in prehospital settings. Hemoglobin-based oxygen carriers (HBOCs) offer both hemodynamic support and oxygen-carrying capacity while avoiding logistical constraints of WB. We hypothesized that low-volume resuscitation of severe hemorrhagic shock with an HBOC (PEGylated carboxyhemoglobin, [PC]) would improve hemodynamic recovery and 72-hour survival; comparable to WB and superior to LR., Materials and Methods: A total of 21 anesthetized male Sprague-Dawley rats underwent severe hemorrhagic shock followed by randomly assigned low-volume resuscitation with LR, WB, or PC, and then recovered from anesthesia for up to 72-hour observation. Mean arterial pressure (MAP) was recorded continuously under anesthesia, and arterial blood gases were measured at baseline (BL), 60 minutes post-hemorrhage (HS1h), and 24 hours post-resuscitation (PR24h). Survival was presented on a Kaplan-Meier plot and significance determined with a log-rank test. Cardiovascular and blood gas data were assessed with one-way analysis of variance and post hoc analysis where appropriate., Results: All measured cardiovascular and blood chemistry parameters were equivalent between groups at BL and HS1h. BL MAP values were 90 ± 3, 86 ± 1, and 89 ± 2 mmHg for LR, PC, and WB, respectively. Immediately following resuscitation, MAP values were 57 ± 4, 74 ± 5, and 62 ± 3 mmHg, with PC equivalent to WB and higher than LR (P < 0.05). WB and LR were both lower than BL (P < 0.0001), whereas PC was not (P = 0.13). The PC group's survival to 72 hours was 57%, which was not different from WB (43%) and higher than LR (14%; P < 0.05)., Conclusions: A single bolus infusion of PC produced superior survival and MAP response compared to LR, which is the standard fluid resuscitant carried by combat medics. PC was not different from WB in terms of survival and MAP, which is encouraging because its reduced logistical constraints make it viable for field deployment. These promising findings warrant further development and investigation of PC as a low-volume, early treatment for hemorrhagic shock in scenarios where blood products may not be available., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Association of Military Surgeons of the United States. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hemolytic anemia (HA) is reduced blood oxygen-carrying capacity resulting from the depletion of red blood cells. Treatment for severe cases involves transfusion to improve oxygen delivery (Do 2 ), which carries risk. In humans, a total hemoglobin (tHb) concentration of 8 g/dL is severe, and <7 g/dL indicates transfusion. Some evidence suggests that compensatory mechanisms maintaining Do 2 are not compromised until <5 g/dL rendering transfusion at 7 g/dL premature. A Sprague-Dawley rat model of phenylhydrazine-induced HA was assessed over decreasing tHb for a Do 2 decompensation point. Three groups (100, 50, or 25% tHb, equating to 16.4, 7.4, or 3.2 g/dL) were generated. Cardiopulmonary, blood chemistry, and oxygenation parameters were measured under anesthesia. Vasoconstrictive responsiveness to phenylephrine was assessed in the exteriorized spinotrapezius. For 50% tHb, cardiopulmonary parameters, Do 2 , and lactate levels were similar to those for 100% tHb. Enhanced vasoconstriction occurred with 50% tHb ( P < 0.0001), not 25% tHb. The 25% group showed decreases in cardiopulmonary parameters, Do 2 , and lactate levels compared with the 100% and 50% groups ( P < 0.05). Do 2 showed a positive correlation with lactate levels at 25% tHb, but decompensation, defined by peripheral hypoxia, was not reached. This is the first study relating Do 2 to tHb in rats. A 50% reduction in tHb was supported by vascular compensation, whereas 25% tHb levied the cardiopulmonary system. A decompensation point was not identified. A rising need for treatment as tHb levels decline below 8 g/dL is evident, but, as compensatory mechanisms remain intact as tHb approaches 3.2 g/dL in rats, a transfusion limit of 5 g/dL in healthy patients is supported. NEW & NOTEWORTHY Early, chronic compensation to severe hemolytic anemia is vascular, switching to cardiopulmonary support as hemoglobin levels decline. Oxygen delivery does not correlate with serum lactate level until total hemoglobin is reduced by 75%.

Background: Hemorrhage is the leading cause of preventable, traumatic death. Currently, prehospital resuscitation fluids provide preload but not oxygen-carrying capacity-a critical blood function that mitigates microvascular ischemia and tissue hypoxia during hemorrhagic shock. Solutions containing polymerized hemoglobin have been associated with vasoactive and hypertensive events. A novel hemoglobin-based oxygen carrier, modified with PEGylation and CO moieties (PEG-COHb), may overcome these limitations., Objectives: To evaluate the systemic and microcirculatory effects of PEG-COHb as compared with the 6% hetastarch in a rat model of hemorrhagic shock., Methods: Male Sprague Dawley rats (N = 20) were subjected to severe, controlled, hemorrhagic shock. Animals were randomized to 20% estimated blood-volume resuscitation with either 6% hetastarch or PEG-COHb. Continuous, invasive, cardiovascular measurements, and arterial blood gases were measured. Microcirculatory measurements of interstitial oxygenation (PISFO2) and vasoactivity helped model oxygen delivery in the spinotrapezius muscle using intravital and phosphorescence quenching microscopy., Results: Hemorrhage reduced mean arterial pressure (MAP), arteriolar diameter, and PISFO2, and increased lactate 10-fold in both groups. Resuscitation with both PEG-COHb and hetastarch improved cardiovascular parameters. However, PEG-COHb treatment resulted in higher MAP (P < 0.001), improved PISFO2 (14 [PEG-COHb] vs. 5 [hetastarch] mmHg; P < 0.0001), lower lactate post-resuscitation (P < 0.01), and extended survival from 90 to 142 min (P < 0.001) as compared with the hetastarch group., Conclusions: PEG-COHb improved MAP PISFO2, lactate, and survival time as compared with 6% hetastarch resuscitation. Importantly, hypertension and vasoactivity were not detected in response to PEG-COHb resuscitation supporting further investigation of this resuscitation strategy.

Decidual macrophages secrete proteases that activate protease-activated receptor 1 (PAR-1). We hypothesized that activation of the inflammatory response by bacteria is amplified by proteases, initiating labor. In addition, we hypothesized that commensal bacteria trigger an inflammatory response by activating NF-κB and TET methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, via a protease amplified PAR-1, RhoA kinase (ROCK) pathway. To evaluate these hypotheses, we compared responses of mononuclear cells with Lactobacillus crispatus, prevalent in the vaginal microbiome of women of European ancestry, with L. iners and Fusobacterium nucleatum, which are more prevalent in vaginal samples collected from African-American women. Decidual tissue was collected at term not-in-labor (TNL), term labor (TL), spontaneous preterm labor (sPTL), and infected preterm labor (iPTL) and immunostained for PAR-1, TET2, and CD14. Mononuclear cells and THP-1 macrophage cells were treated with bacteria and elastase, a known activator of PAR-1. The inflammatory response was monitored by confocal microscopy of TET2 and the p65 subunit of NF-κB, as well as IL-8 production. Decidual staining for PAR-1, TET2, and CD14 increased TNL < TL < sPTL < iPTL. All treatments stimulated translocation of TET2 and p65 from the cytosol to the nucleus and increased IL-8, but L. iners and F. nucleatum caused more robust responses than L. crispatus. Inhibition of PAR-1 or ROCK prevented TET2 and p65 nuclear translocalization and increases in IL-8. Our findings demonstrate that proteases amplify the inflammatory response to commensal bacteria. The more robust response to bacteria prevalent in African-American women may contribute to racial disparities in preterm birth.

Hypoxia drives sickle cell disease (SCD) by inducing sickle cell haemoglobin to polymerize and deform red blood cells (RBC) into the sickle shape. A novel carboxyhaemoglobin-based oxygen carrier (PEG-COHb; PP-007) promotes unsickling in vitro by relieving RBC hypoxia. An in vivo rat model of vaso-occlusive crisis (VOC) capable of accommodating a suite of physiological and microcirculatory measurements was used to compare treatment with PEG-COHb to a non-oxygen carrying control solution (lactated ringer's [LRS]). Male Sprague-Dawley rats were anesthetized and surgically prepared to monitor microvascular interstitial oxygenation (P ISF O 2 ), cardiovascular parameters and blood chemistry. Human homozygous SCD RBCs were isolated and exchange transfused into the rats until the distal microcirculation of the exteriorized spinotrapezius muscle was hypoxic and RBC aggregates were visualized. VOC was left untreated (Sham) or treated 15 min later with PEG-COHb or LRS and observed for up to 4 h. Treatment with PEG-COHb showed better improvement of P ISF O 2 , end-point lactate, mean arterial pressure and survival duration compared to Sham and LRS. Restoring P ISF O 2 was associated with relieving the RBC aggregates driving VOC, which then affected other study metrics. Compared to LRS, PEG-COHb's oxygen-carrying properties were key to improved outcomes.

Background: Hemorrhage and its complications are the leading cause of preventable death from trauma in young adults, especially in remote locations. To address this, deliverable, shelf-stable resuscitants that provide therapeutic benefits throughout the time course of hemorrhagic shock and the progressive ischemic injury it produces are needed. SANGUINATE is a novel bovine PEGylated carboxyhemoglobin-based oxygen carrier, which has desirable oxygen-carrying and oncotic properties as well as a CO moiety to maintain microvascular perfusion., Objectives: To compare the crystalloid (Lactated Ringer's Solution; LRS), and the colloid (Hextend) standards of care with SANGUINATE in a post "golden hour" resuscitation model., Methods: Rats underwent a controlled, stepwise blood withdrawal (45% by volume), were maintained in untreated hemorrhagic shock state for >60 min, resuscitated with a 20% bolus of one of the three test solutions, and observed till demise. Parameters of tissue oxygenation (PISFO2), arteriolar diameters, and mean arterial pressure (MAP) were collected., Results: SANGUINATE-treated animals survived significantly longer than those treated with Hextend and LRS. SANGUINATE also significantly increased tissue PISFO2 2 h after resuscitation, whereas LRS and Hextend did not. SANGUINATE also produced a significantly higher MAP, which was hypotensive compared to baseline, that endured until demise., Conclusions: Resuscitation with SANGUINATE after prolonged hemorrhagic shock improves survival, MAP, and PISFO2 compared with standard of care plasma expanders. Since the pathologies of hemorrhagic shock and the associated systemic ischemia are progressive, preclinical studies of this nature are essential to determine efficacy of new resuscitants across the range of possible times to treatment.

Ox66™ is a novel solid state oxygenating compound. In order to support the use of Ox66™ as a potential oxygenating supplement to injured cells, this study evaluated the safety of Ox66™, its ability to withstand the conditions in the digestive tract, and its potential to increase oxygenation in the mesentery in rats. The toxicity of Ox66™ was evaluated by performing acute (10-day) and chronic (90-day) feeding studies on rats, the stability of the compound in the digestive tract was evaluated via ex vivo simulated digestion and subsequent CFDA viability assay on gut epithelial cells, and its capacity for oxygenation in the mesenteric microcirculation was determined by interstitial fluid pressure (P ISF ) O 2 measurements upon injection into the small intestine of rats. No toxicity was found associated with acute or chronic oral administration of the compound in rats, and the compound was able to withstand the environment of the digestive tract in vitro. Based on the acute animal feeding study, the NOAEL was considered to be 1000 mg/kg/day. This proof-of-concept study further demonstrates the potential of Ox66™ to function as an oxygenating supplement that might be useful for treating either pathological hypoxic-related conditions or to improve oxygenation levels during or after exercise under healthy conditions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinase-1 (MMP-1) and neutrophil elastase are proteolytic enzymes involved in tissue remodeling, but a role for them as uterotonic agents has not been considered. However, both these proteases activate protease-activated receptor 1 (PAR-1) that mediates thrombin-induced contractions. Matrix metalloproteinase-1 and elastase are products of neutrophils that infiltrate intrauterine tissues at the time of labor, so we tested the hypothesis that these proteases might be novel uterotonic agents acting via PAR-1. Decidual tissue was collected from fetal membranes of term not-in-labor (TNL), term labor (TL), and preterm labor (PTL) women and analyzed for gene and protein expression of MMP-1 and neutrophil elastase. Contractile effects of MMP-1 and elastase were tested with uterine strips of day 19 and 20 gestation rats. Expression of MMP-1 and neutrophil elastase was increased in TL and PTL as compared to TNL. Expression of both the pro- and active enzymes of MMP-1 increased progressively from TNL to TL to PTL. Tumor necrosis factor-α, a neutrophil product, increased MMP-1 in decidual and myometrial cells. Both MMP-1 and elastase stimulated strong contractions of myometrial strips, which were prevented by inhibition of PAR-1 and inhibition of inositol trisphosphate receptor or calcium channel blockade. Indomethacin did not prevent protease-induced contractions. These data suggest that MMP-1 and neutrophil elastase may be important but heretofore unrecognized players in stimulating uterine contractions at the time of labor, and they may explain why indomethacin delays, but does not prevent, PTL because indomethacin inhibits the prostaglandin component but not the protease component of labor.

Under physiologic conditions, microvascular oxygen delivery appears to be well matched to oxygen consumption in respiring tissues. We present a technique to measure interstitial oxygen tension (PISFO2) and oxygen consumption (VO2) under steady-state conditions, as well as during the transitions from rest to activity and back. Phosphorescence Quenching Microscopy (PQM) was employed with pneumatic compression cycling to achieve 1 to 10 Hz sampling rates of interstitial PO2 and simultaneous recurrent sampling of VO2 (3/min) in the exteriorized rat spinotrapezius muscle. The compression pressure was optimized to 120-130 mmHg without adverse effect on the tissue preparation. A cycle of 5s compression followed by 15s recovery yielded a resting VO2 of 0.98 ± 0.03 ml O2/100 cm(3)min while preserving microvascular oxygen delivery. The measurement system was then used to assess VO2 dependence on PISFO2 at rest and further tested under conditions of isometric muscle contraction to demonstrate a robust ability to monitor the on-kinetics of tissue respiration and the compensatory changes in PISFO2 during contraction and recovery. The temporal and spatial resolution of this approach is well suited to studies seeking to characterize microvascular oxygen supply and demand in thin tissues., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Matrix metalloproteinase 1 (MMP-1) is an activator of protease-activated receptor 1 (PAR-1), which is known to mediate the release of endothelin 1 (ET-1) in endothelial cells and activate the RhoA kinase (ROCK) pathway. Recently, we reported increased serum and vascular MMP-1 in women with preeclampsia and hypothesized that the action of MMP-1 on PAR-1 might have vasoconstrictive effects. Resistance-sized omental arteries obtained from normal pregnant women were mounted on a myograph system and perfused with MMP-1 in a dose range of 0.025 to 25 ng/mL or with angiotensin II (Ang II) in a dose range of 0.001 to 10 µmol/L in the presence of intraluminal MMP-1 (2.5 ng/mL) perfusion. Angiotensin II dose response was also performed with omental arteries from women with preeclampsia. Matrix metalloproteinase 1 caused dose-dependent vasoconstriction in endothelium-intact, but not in endothelium-denuded, vessels from normal pregnant women, which was blocked by inhibitors of PAR-1 and ET-1 type A receptor blocker. Intraluminal perfusion with a constant amount of MMP-1 enhanced vessel reactivity to Ang II, which was blocked by inhibitors of PAR-1, ROCK, and ET-1. Enhanced vascular reactivity to Ang II was observed in endothelium-intact, but not in endothelium-denuded, arteries of women with preeclampsia. Inhibitors of PAR-1, ROCK, and ET-1 blocked enhanced vascular reactivity to Ang II in endothelium-intact preeclamptic arteries. These data demonstrate that MMP-1 has potent vasoconstrictor effects and the ability to enhance vascular reactivity to vasoconstrictor hormones, which are mediated by an endothelial PAR-1, ROCK, and ET-1 pathway. Increased circulating levels of MMP-1 and its increased expression in systemic vessels of women with preeclampsia may contribute to the development of maternal hypertension., (© The Author(s) 2015.)

Pyoderma gangrenosum (PG) is a neutrophil-predominant inflammatory disease that initially presents as a sterile pustule and may progress to ulcerations. Its root cause is unknown, but the presentation is commonly associated with systemic inflammatory conditions such as inflammatory bowel disease, arthritis and haematological abnormalities. On the other hand, pregnant women show a progressive neutrophilia during gestation, which culminates in a major inflammatory event to help drive labour. Although uncommonly, PG has been associated with pregnancy, which provides an additional link to systemic inflammation as an underlying cause of PG. We reviewed documented presentations of PG in gravid and post-partum patients, and have speculated on the possible pathogenesis based on their clinical presentations. Also, we summarize the reported treatments and their outcomes in these patients., (© 2015 British Association of Dermatologists.)

The effects of a polymerized bovine hemoglobin-based oxygen carrier (HBOC) and two derivatives on arteriolar vasoactivity and tissue oxygen tension were explored by administering HBOC in a dose-response fashion to normovolemic rats. The effect of oxygen affinity (P50) and viscosity was also explored, where the P50 and viscosity of the parent compound (HBOC-201) and its modifications (MP50 and LP50A) were as follows: 40mmHg and 3.0cP (HBOC-20l); 18mmHg and 4.4cP (MP50); and 17mmHg and 12.1cP (LP50A). Anesthetized male Sprague-Dawley rats (N=32) were randomized to receive one of the HBOC solutions, and were administered four infusions that increased in concentration for each dose (2, 22, 230 and 780mg/kg, IV). Data were compared to rats receiving an equivalent volume for each of the four infusions (0.4, 0.4, 3.8, 13.1ml/kg, IV) of iso-oncotic 5.9% human serum albumin (HSA). Increasing doses of either HBOC solutions or HSA were associated with increasing MAP. Doses 3 and 4 of HBOC-201, MP50 and HSA produced significant increases in MAP, whereas similar increases began at a lower dose (Dose 2) with LP50A. There were no significant changes in arteriolar diameters at any dose for any group. Interstitial partial pressure of oxygen (ISF PO2) remained unchanged for HBOC-201, MP50 and HSA, but LP50A caused a significant decrease in ISF PO2 compared to baseline after Doses 3 and 4. In conclusion, there was no evidence that HBOC-201 would perform better with increased oxygen affinity (40 to 18mmHg) or viscosity (3.0 to 4.4cP)., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study was designed to test the effect of top-load infusions of increasing doses of two versions of the novel, high molecular weight hemoglobin-based oxygen carrier, OxyVita and OxyVita C solution ([Hb] = 6 g/dL), on mean arterial pressure (MAP), arteriolar diameter, and tissue oxygenation. Experiments were carried out on 18 anesthetized male Sprague-Dawley rats in which microcirculatory observations were made on the spinotrapezius muscle. Intravenous infusions of four increasing doses of the OxyVita solutions (2, 22, 230, and 780 mg/kg) were made for each group, and a separate group of animals was used for volume control. Tissue oxygenation was measured as interstitial fluid (ISF) PO2 using phosphorescence quenching microscopy. Increasing doses of either OxyVita solution or Lactated Ringer's solution (LRS, volume control) were associated with increasing MAP. For LRS infusions, MAP returned to baseline between each incremental dose injected, whereas there was an incomplete return for either of the OxyVita solutions. ISF PO2 for OxyVita was significantly lower than that for either LRS or OxyVita C, whereas ISF PO2 for OxyVita C was never statistically different from LRS. There were no significant changes in arteriolar diameters for LRS and either of the OxyVita solutions., (Reprint & Copyright © 2013 Association of Military Surgeons of the U.S.)

Preeclamptic women have enhanced blood pressure response to angiotensin II and extensive systemic vascular infiltration of neutrophils. Neutrophils release reactive oxygen species that might activate the RhoA kinase pathway to enhance vascular reactivity. We hypothesized that enhanced vascular reactivity in preeclampsia is attributed to neutrophil-mediated reactive oxygen species activation of the RhoA kinase pathway. Omental arteries were obtained at cesarean section and studied using a myograph system. We found that arteries of preeclamptic women had extensive infiltration of neutrophils and enhanced reactivity to angiotensin II. Treatment of arteries of normal pregnant women with reactive oxygen species or activated neutrophils enhanced vessel reactivity to angiotensin II mimicking preeclamptic vessels. Pretreatment with superoxide dismutase/catalase to quench reactive oxygen species or RhoA kinase inhibitor blocked enhanced responses in preeclamptic and normal vessels. Reactive oxygen species also enhanced vessel reactivity to norepinephrine, which was blocked by RhoA kinase inhibition. Treatment of arteries with reactive oxygen species increased RhoA kinase activity 3-fold, whereas culture of human vascular smooth muscle cells with angiotensin II and activated neutrophils or reactive oxygen species resulted in phosphorylation of key proteins in the RhoA kinase pathway. We conclude that enhanced vascular reactivity of omental arteries in preeclampsia is attributed to reactive oxygen species activation of the RhoA kinase pathway and that enhanced vascular reactivity is likely attributed to the infiltration of neutrophils. We speculate that neutrophil infiltration into systemic vasculature of preeclamptic women is an important mechanism for hypertension.