Cell-free oxidized hemoglobin drives reactive oxygen species production and pro-inflammation in an immature primary rat mixed glial cell culture.

BACKGROUND: Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with deposition of redox active cell-free hemoglobin (Hb), derived from hemorrhagic cerebrospinal fluid (CSF), in the cerebrum and cerebellum. In a recent study, using a preterm rabbit pup model of IVH, intraventricularly administered haptoglobin (Hp), a cell-free Hb scavenger, partially reversed the damaging effects observed following IVH. Together, this suggests that cell-free Hb is central in the pathophysiology of the injury to the immature brain following GM-IVH. An increased understanding of the causal pathways and metabolites involved in eliciting the damaging response following hemorrhage is essential for the continued development and implementation of neuroprotective treatments of GM-IVH in preterm infant. METHODS: We exposed immature primary rat mixed glial cells to hemorrhagic CSF obtained from preterm human infants with IVH (containing a mixture of Hb-metabolites) or to a range of pure Hb-metabolites, incl. oxidized Hb (mainly metHb with iron in Fe3+), oxyHb (mainly Fe2+), or low equivalents of heme, with or without co-administration with human Hp (a mixture of isotype 2-2/2-1). Following exposure, cellular response, reactive oxygen species (ROS) generation, secretion and expression of pro-inflammatory cytokines and oxidative markers were evaluated. RESULTS: Exposure of the glial cells to hemorrhagic CSF as well as oxidized Hb, but not oxyHb, resulted in a significantly increased rate of ROS production that positively correlated with the rate of production of pro-inflammatory and oxidative markers. Congruently, exposure to oxidized Hb caused a disintegration of the polygonal cytoskeletal structure of the glial cells in addition to upregulation of F-actin proteins in microglial cells. Co-administration of Hp partially reversed the damaging response of hemorrhagic CSF and oxidized Hb. CONCLUSION: Exposure of mixed glial cells to oxidized Hb initiates a pro-inflammatory and oxidative response with cytoskeletal disintegration. Early administration of Hp, aiming to minimize the spontaneous autoxidation of cell-free oxyHb and liberation of heme, may provide a therapeutic benefit in preterm infant with GM-IVH.

Efficacy of resuscitative infusion with hemoglobin vesicles in rabbits with massive obstetric hemorrhage.

BACKGROUND: Hemoglobin vesicles have been developed as artificial oxygen carriers, and they have the potential to serve as a substitute for red blood cell transfusion. OBJECTIVE: This study aimed to evaluate the efficacy of hemoglobin vesicle infusion for the initial treatment instead of red blood cell transfusion in rabbits with massive obstetric hemorrhage. STUDY DESIGN: Pregnant New Zealand white rabbits (28th day of pregnancy; normal gestation period, 29-35 days) underwent uncontrolled hemorrhage to induce shock by transecting the right midartery and concomitant vein in the myometrium. Subsequently, rabbits received isovolemic fluid resuscitation through the femoral vein with an equivalent volume of hemorrhage every 5 minutes. Resuscitative infusion regimens included 5% human serum albumin (n=6), stored washed red blood cells with plasma (vol/vol=1:1; n=5), and hemoglobin vesicle with 5% human serum albumin (vol/vol=4:1; n=5). A total of 60 minutes after the start of bleeding, rabbits underwent surgical hemostasis by ligation of the bleeding vessels and then were monitored for survival within 24 hours. RESULTS: During fluid resuscitation, hemoglobin vesicle infusion and red blood cell transfusion maintained a mean arterial pressure of >50 mm Hg and a hemoglobin concentration of >9 g/dL and prevented the elevation of plasma lactate. In contrast, resuscitation with 5% human serum albumin alone could not prevent hemorrhagic shock as evidenced by a low mean arterial pressure (40 mm Hg), a low hemoglobin concentration (2 g/dL), and a marked elevation of plasma lactate. All animals in the red blood cell group and the hemoglobin vesicle group survived more than 8 hours, whereas all animals in the 5% human serum albumin group died within 8 hours. CONCLUSION: Hemoglobin vesicle infusion may be effective in the initial management of massive obstetric hemorrhage.

Carbon Monoxide-Saturated Polymerized Placenta Hemoglobin Optimizes Mitochondrial Function and Protects Heart Against Ischemia-Reperfusion Injury.

ABSTRACT: Ischemia-reperfusion (I-R) injury is detrimental to cardiovascular system. This study was designed to investigate whether carbon monoxide-saturated polymerized human placenta hemoglobin (CO-PolyPHb) attenuates cardiac I-R injury and to elucidate the underlying mechanism(s). Sixty male adult Sprague-Dawley rats were randomly divided into 6 groups: saline + sham group, PolyPHb + sham group, CO-PolyPHb + sham group, saline + I-R group, PolyPHb + I-R group, and CO-PolyPHb + I-R group. Rats were pretreated with injection of PolyPHb, CO-PolyPHb (0.5 g Hb/kg/d), or an equivalent volume of saline via caudal vein for 3 days. After pretreatment, hearts were isolated Langendorff perfused and subjected to 30-minute no-flow ischemia and 120-minute reperfusion. As compared with the saline + I-R group, pretreatment with CO-PolyPHb greatly improved the recovery of cardiac function, reduced infarct size, and suppressed the release of cardiac enzyme. Importantly, CO-PolyPHb showed more prominent cardioprotective effect than PolyPHb, exhibiting a promising therapeutic potential in cardiac I-R injury. Further study demonstrated that CO-PolyPHb activated molecular signaling toward mitophagy and significantly elevated the mitochondrial respiratory function in the heart. In addition, CO-PolyPHb upregulated the phosphorylation of the proteins in insulin signaling pathway and increased the glucose uptake rate in cardiomyocytes. Pharmacological inhibition of this pathway by wortmannin abrogated the anti-I-R effect of CO-PolyPHb. In conclusion, using an isolated rat heart model, we have demonstrated that pretreatment with CO-PolyPHb provided protective effect against cardiac I-R injury, and this protection was mediated by the improvement of mitochondrial function and activation of insulin signaling pathway in the heart.

Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury.

Traumatic brain injury (TBI) represents a major cause of death and disability worldwide. Exacerbated neuroinflammation following TBI causes secondary injury. Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein that promotes the inflammatory response in different tissues and cells. However, the contribution of PDPN to neuroinflammation and microglial activation is unknown. Here, we found that PDPN was correlated with microglial activation after TBI in mice. Meanwhile, PDPN expression could be induced by trauma-related stimuli, such as lipopolysaccharide (LPS), ATP, H2O2 and hemoglobin (Hb), in primary microglia. Furthermore, with Hb treatment in vitro, knockdown of PDPN could decrease the proportion of M1-like microglia and increase the proportion of M2-like microglia via reduced secretion of IL-1ß and TNF-α and increased secretion of IL-10 and TGF-ß compared to the control microglia. Immunofluorescence also showed that CD86-positive microglia were decreased and CD206-positive microglia were elevated in the PDPN-KD group. Additionally, PDPN knockdown impaired microglial mobility and phagocytosis and decreased the expression of matrix metalloproteinases (mainly MMP2 and MMP9). In summary, PDPN plays an important role in microglia-mediated inflammation and may serve as a potential target for TBI treatment.

A case study of 10 patients administered HBOC-201 in high doses over a prolonged period: outcomes during severe anemia when transfusion is not an option.

BACKGROUND: Hemoglobin-Based Oxygen Carriers (HBOCs) can act as an "oxygen bridge" in acute severe anemia when transfusion is indicated, but not possible. We present data on 10 Expanded Access (EA) patients treated with high cumulative doses of Hemopure (HBOC-201), to assess the ability of HBOC-201 to safely treat life threatening anemia in situations where high volumes of product were administered over an extended period of time. STUDY DESIGN AND METHODS: Inclusion in this study required that the patient receive at least 10 units of HBOC-201 between 2014 and 2017 under the FDA-sanctioned EA program. Depending on a patient's geographical location, treatment with HBOC-201 was obtained through either a single patient emergency Investigational New Drug (IND) application, or an intermediate size population IND. Of the 41 patients who were treated during this period, 10 patients received 10 or more units of the product. Data were obtained from medical records. RESULTS: Treatments with HBOC-201 started within 24 hours of signing consent and were administered at an average rate of 1.99 (SD 0.17) units per day over a mean of 8.2 days (SD 2.9), during which patients received on average 16.2 units (SD 5.7 units) of HBOC-201. The median pre-treatment nadir corpuscular hemoglobin (Hb) concentration was 3.3 (SD 0.9) g/dL and post-treatment Hemoglobin was 7.3 (SD 1.7) g/dL. Common side effects included methemoglobinemia, gastrointestinal symptoms, and hypertension. However, no product-related serious adverse events (SAEs) were noted. All patients survived. CONCLUSIONS: Administration of HBOC-201 over an extended period is a feasible and safe oxygen bridge for severely anemic patients who cannot be transfused with RBC.

Structure-activity relationship study on new hemorphin-4 analogues containing steric restricted amino acids moiety for evaluation of their anticonvulsant activity.

In the present study, several new analogues of hemorphin-4, modified with unnatural conformationally restricted amino acids followed the structure Aaa-Tyr-Xxx-Trp-Thr-NH2, where Aaa is the low-molecular-weight lipophilic adamantyl building block, and Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) was synthesized, characterized and investigated for anticonvulsant activity in three seizure tests, the maximal electroshock test (MES), 6-Hz psychomotor seizure test and timed intravenous pentylenetetrazole infusion (ivPTZ) test. The acute neurological toxicity was determined using the rota-rod test. The new synthetic neuropeptide analogues were prepared by solid-phase peptide synthesis-Fmoc chemistry and were evaluated in three doses of 1, 3 and 5 µg, respectively, administered intracerebroventricularly in male ICR mice. The physicochemical properties of these peptide analogues were evaluated as pKa and pI values were calculated using potentiometry. The IR spectrum of the compounds was recorded and the characteristic lines of both adamantane moiety and the peptide backbone were registered in the wavelength range from 4000 to 400 cm-1. The hexapeptide Ang IV was used as a positive control. From the six synthesized peptide analogues, the P4-5 was the most active at doses of 1 and 3 µg in the three seizure tests. The order of potency of other peptides was as follows: P4 > P4-3 = P4-4 > P4-2 > Ang IV in MES, P4-4 ≥ P4-1 > P4-3 > P4-2 > P4 > Ang IV in 6-Hz test and P4-4 = P4-3 > P4-2 = P4 > Ang IV in ivPTZ test. None of the peptides displayed neurotoxicity in the rota-rod test. Docking study results suggest that direct H-bonding and ionic interactions between our synthetic ligands and residues, responsible for coordination of Zn2+ along with hydrophobic interactions between our ligands and IRAP active site are the most important for the ligand binding. The results propose that incorporation of adamantane and cycloalkane building blocks in the peptide chain of the hemorphin-4 scaffold is important for the potential high biological activity.

Pharmaceutical Technology Innovation Strategy Based on the Function of Blood Transport Proteins as DDS Carriers for the Treatment of Intractable Disorders and Cancer.

Blood transport proteins are biogenic molecules with unique and interesting inherent characteristics that make up living organisms. As the utilization of their inherent characteristics can be a groundbreaking strategy to resolve and improve several clinical problems, attempts have been made to develop pharmaceutical and biomedical preparations based on blood transport proteins for the treatment and diagnosis of disorders. Among various blood transport proteins, we focus on the immense potential of hemoglobin and albumin to serve as carriers of biomedical gases (oxygen and carbon monoxide) and anticancer agents (low-molecular compounds and antisense oligodeoxynucleotides), respectively, for the development of innovative drug delivery systems (DDS) to treat intractable disorders and solid cancers. In this review, I introduce the pharmaceutical technology, strategies, and application of DDS carriers that have been designed on the basis of the structure and function of hemoglobin and albumin. In addition, the prospect of using hemoglobin and albumin as materials for DDS carriers is discussed.

Continuous hemoglobin and plethysmography variability index monitoring can modify blood transfusion practice and is associated with lower mortality.

To determine the effect of implementing an algorithm of fluid and blood administration based on continuous monitoring of hemoglobin (SpHb) and PVI (plethysmography variability index) on mortality and transfusion on a whole hospital scale. This single-center quality program compared transfusion at 48 h and mortality at 30 days and 90 days after surgery between two 11-month periods in 2013 and 2014 during which all the operating and recovery rooms and intensive care units were equipped with SpHb/PVI monitors. The entire team was trained to use monitors and the algorithm. Team members were free to decide whether or not to use devices. Each device was connected to an electronic wireless acquired database to anonymously acquire parameters on-line and identify patients who received the monitoring. All data were available from electronic files. Patients were divided in three groups; 2013 (G1, n = 9285), 2014 without (G2, n = 5856) and with (G3, n = 3575) goal-directed therapy. The influence of age, ASA class, severity and urgency of surgery and use of algorithm on mortality and blood use were analyzed with cox-proportional hazard models. Because in 2015, SpHb/PVI monitors were no longer available, we assessed post-study mortality observed in 2015 to measure the impact of team training to adjust vascular filling on a patient to patient basis. During non-cardiac surgery, blood was more often transfused during surgery in G3 patients as compared to G2 (66.6% vs. 50.7%, p < 0.001) but with fewer blood units per patient. After adjustment, survival analysis showed a lower risk of transfusion at 48 h in G3 [OR 0.79 (0.68-0.93), p = 0.004] but not in G2 [OR 0.90 (0.78-1.04) p = 0.17] as compared to G1. When adjusting to the severity of surgery as covariable, there was 0.5 and 0.7% differences of mortality at day 30 and 90 whether patients had goal directed therapy (GDT). After high risk surgery, the mortality at day 30 is reduced by 4% when using GDT, and 1% after intermediate risk surgery. There was no difference for low risk surgery. G3 Patients had a lower risk of death at 30 days post-surgery [OR 0.67 (0.49-0.92) p = 0.01] but not G2 patients [OR 1.01, (0.78-1.29), p = 0.96]. In 2015, mortality at 30 days and 90 days increased again to similar levels as those of 2013, respectively 2.18 and 3.09%. Monitoring SpHb and PVI integrated in a vascular filling algorithm is associated with earlier transfusion and reduced 30 and 90-day mortality on a whole hospital scale.

Implementation of a Nurse-Empowered Renal Anemia Protocol in Children.

A high proportion of patients on hemodialysis persist with low hemoglobin levels despite receiving treatment with erythropoiesis-stimulating agents. A registered nurse-driven renal anemia protocol was designed and implemented by a team in a pediatric hemodialysis unit. We compared proportion of patients achieving the target hemoglobin (Hgb) and transferrin saturation (TSAT) before and after the implementation of the protocol. There was an increase in patients achieving the target Hgb and TSAT range, with an increase in the Hgb concentration. There were no differences in the proportion of patients with left ventricular hypertrophy, erythropoiesis-stimulating agents or intravenous iron dose, transfusion rates, or hospitalization rates. The implementation of a nurse-driven anemia protocol in a pediatric hemodialysis unit increased the proportion of patients achieving target Hgb and TSAT range without a rise in medication doses.

Anti-N and anti-Doa immunoglobulin G alloantibody-mediated delayed hemolytic transfusion reaction with hyperhemolysis in sickle cell disease treated with eculizumab and HBOC-201: case report and review of the literature.

BACKGROUND: Delayed hemolytic transfusion reaction (DHTR) with hyperhemolysis is a potentially fatal complication resulting from alloimmunization that can cause severe hemolysis of both transfused and intrinsic red blood cells (RBCs). Patients with sickle cell disease often receive multiple RBC units during their lifetime and thus are likely to develop alloantibodies that increase the risk for DHTR. Treatment to decrease hemolysis includes intravenous immunoglobulin (IVIG), steroids, eculizumab, rituximab, and plasmapheresis in addition to erythropoietin (EPO), intravenous (IV) iron, vitamin B12, and folate to support erythropoiesis. RBC transfusion is preferably avoided in DHTR due to an increased risk of exacerbating the hemolysis. CASE REPORT: We report a rare case of anti-N and anti-Doa immunoglobulin (Ig)G alloantibody-mediated life-threatening DHTR with hyperhemolysis in a patient with hemoglobin SS after RBC transfusion for acute chest syndrome who was successfully treated with eculizumab and HBOC-201 (Hemopure) in addition to steroids, IVIG, EPO, IV iron, and vitamin B12. HBOC-201 (Hemopure) was successfully used as a RBC alternative in this patient. CONCLUSION: Anti-N and anti-Doa IgG alloantibodies can rarely cause severe life-threatening DHTR with hyperhemolysis. HBOC-201 (Hemopure) can be a lifesaving alternative in this scenario. Our report also supports the use of eculizumab in DHTR; however, prospective studies are needed to determine the appropriate dose and sequence of eculizumab administration.

Wuzhi capsule and haemoglobin influence tacrolimus elimination in paediatric kidney transplantation patients in a population pharmacokinetics analysis: A retrospective study.

WHAT IS KNOWN AND OBJECTIVES: Tacrolimus is widely used for kidney transplantation in children. However, the narrow therapeutic window and considerable interindividual and intraindividual variabilities make tacrolimus untoward to design an optimum dosage for paediatric personalized medicine. Our research aims to establish the tacrolimus population pharmacokinetics (PPK) of Chinese paediatric kidney transplantation patients and to distinguish covariates impacting variabilities. METHODS: Chinese paediatric kidney transplantation patients treated with tacrolimus between January 2014 and April 2018 from Children's Hospital of Fudan University were retrospectively analysed. A total of 51 Chinese paediatric kidney transplantation patients were analysed using non-linear mixed effects modelling (NONMEM). The effects of population characteristics, biological features and drug combination were assessed. The final PPK model was evaluated using visual inspection of routine diagnostic plots and the internal validation method of bootstrap. RESULTS: Our data met the condition of a one-compartment model, and the final model was CL/F = 32.7 × (WT/70)0.75  × (1 - WZ × 0.341) × (HGB/97)-0.508 ; V/F = 1890 × (WT/70) × (POD/57)0.816 , where WT, WZ, HGB and POD were weight, Wuzhi capsule (extracted from schisandra sphenanthera, whose primary efficient constituents are schisantherin A, schisandrol B, schisandrin etc, and often used to treat drug-induced hepatitis in Chinese organ transplant patients), haemoglobin and post-transplant day, respectively. WHAT IS NEW AND CONCLUSION: The tacrolimus PPK model in Chinese paediatric kidney transplantation patients was developed, and Wuzhi capsule and haemoglobin influence tacrolimus elimination in paediatric kidney transplantation patients.

Topical application of haemoglobin: a safety study.

OBJECTIVE: Ischaemia is one of the biggest problems in wound healing. It causes chronic wounds and also prevents normal wound healing because the tissue is oxygen deprived. Most oxygen-supplying therapies are only feasible in a clinical setting, but topical haemoglobin applications, such as Granulox, can be used in a non-clinical setting. For home application, the haemoglobin solution is sprayed topically onto the wound using a pressurised ready-to-use device with a bag-on-valve system. Although this system does not mix product and propellant, the risk of product inhalation by the patient, user or bystanders has to be minimised. This safety study aimed to determine particle size and product concentration in the surroundings after application to determine if there is a risk that product particles enter the respiratory tract. METHODS: Measurements were performed using a laser scattered light photometer and a Scanning Mobility Particle Sizer (SMPS)-Spectrometer at different distances from the measuring devices to determine the inhalation risk for a possible user, patient and bystander. At all measuring points the amount of particles, their size and the formation of dust were measured. RESULTS: No nanoparticles or dust were created during the application of the haemoglobin spray. The concentrations of the measured particles are below the allowed limits defined by Austrian law. CONCLUSION: There is no risk of inhaling nanoparticles or being exposed to harmful concentrations of larger particles of the tested product. All the product's ingredients can be degraded and excreted by the human body through natural pathways.

Cost-effectiveness of adjunct haemoglobin spray in the treatment of hard-to-heal wounds in a UK NHS primary care setting.

OBJECTIVE: To evaluate the cost-effectiveness of topical haemoglobin spray as adjunct therapy in the treatment of hard-to-heal wounds within a UK National Health Service (NHS) community setting. METHOD: In a previously published comparative clinical evaluation, 50 consecutive patients treated with topical haemoglobin spray, as adjunct to standard care and followed up over 26 weeks, were compared with 50 consecutive retrospective controls from the same clinic treated with the same standard care protocol in the year prior to the introduction of adjunct topical haemoglobin spray. A de novo cost-effectiveness and break-even analysis were performed, using data from the previously published clinical evaluation, for all patients (intent-to-treat) and for patients with complete follow-up using a micro-costing approach and considering only wound care dressing costs. RESULTS: At 26 weeks, the total cost of dressings for all patients in the intervention group was £6953 with 874 cumulative weeks healed, compared with £9547 with 278 cumulative weeks healed for all patients in the control group. The incremental cost-effectiveness ratio (ICER), the incremental cost per additional week healed with adjunct topical haemoglobin spray, is therefore negative (dominant). Total treatment costs per week were lower from week six onwards, with break-even estimated to be at week 10.2. When considering only patients with complete follow-up, the results were similarly dominant, with a mean 10.9 more weeks healed, a mean dressing cost saving per patient of £81.83 by week 26 (-37%). Cost savings were realised from week five, and a break-even was estimated to occur at week 8.0. CONCLUSION: Topical haemoglobin spray has the potential to restore the healing process, reduce healing times and reduce dressing costs in a NHS community setting, within a few weeks of adoption.

When might transferrin, hemopexin or haptoglobin administration be of benefit following the transfusion of red blood cells?

PURPOSE OF REVIEW: After transfusion, a percentage of red blood cells undergo hemolysis within macrophages. Intravascular exposures to hemin and hemoglobin (Hb) can occur after storage bag hemolysis, some transfusion reactions, during use of medical assist devices and in response to bacterial hemolysins. Proteins that regulate iron, hemin and Hb either become saturated after iron excess (transferrin, Tf) or depleted after hemin (hemopexin, Hpx) and Hb (haptoglobin, Hp) excess. Protein saturation or stoichiometric imbalance created by transfusion increases exposure to non-Tf bound iron, hemin and Hb. Tf, Hpx and Hp are being developed for hematological disorders where iron, hemin and Hb contribute to pathophysiology. However, complexed to their ligands, each represents a potential iron source for pathogens, which may complicate the use of these proteins. RECENT FINDINGS: Erythrophagocytosis by macrophages and processes of cell death that lead to reactive iron exposure are increasingly described. In addition, the effects of transfusion introduced circulatory hemin and Hb are described in the literature, particularly following large volume transfusion, infection and during concomitant medical device use. SUMMARY: Supplementation with Tf, Hpx and Hp suggests therapeutic potential in conditions of extravascular/intravascular hemolysis. However, their administration following transfusion may require careful assessment of concomitant disease.

Novel Hemoglobin-Based Oxygen Carrier Bound With Albumin Shows Neuroprotection With Possible Antioxidant Effects.

Background and Purpose- A hemoglobin-albumin cluster, 1 core of hemoglobin covalently bound with 3 shell albumins, designated as HemoAct was developed as a hemoglobin-based oxygen carrier. We aim to investigate neuroprotection by HemoAct in transient cerebral ischemia and elucidate its underlying mechanisms. Methods- Male rats were subjected to 2-hour transient middle cerebral artery occlusion and were then administered HemoAct transarterially at the onset of reperfusion. Neurological and pathological findings were examined after 24 hours of reperfusion to identify neuroprotection by HemoAct. Intermittent measurements of cortical blood flow and oxygen content were performed, and a histopathologic analysis was conducted on rats during the early phase of reperfusion to assess the therapeutic mechanism of HemoAct. In addition, the antioxidant effects of HemoAct were examined in hypoxia/reoxygenation-treated rat brain microvascular endothelial cells. Results- Neurological deterioration, infarct and edema development, and the activation of MMP-9 (matrix metalloprotease-9) and lipid peroxidation after 24 hours of reperfusion were significantly ameliorated by the HemoAct treatment. Reductions in blood flow and tissue partial oxygen pressure in the cortical penumbra after 6 hours of reperfusion were significantly ameliorated by the HemoAct treatment. The histopathologic analysis of the cortical penumbra revealed that HemoAct in HemoAct-treated rats showed superior microvascular perfusion with the mitigation of microvascular narrowing changes than autologous erythrocytes in nontreated rats. Although HemoAct extravasated into the ischemic core with serum protein, it did not induce an increase in serum extravasation or reactive oxygen species production in the ischemic core. In vitro experiments with rat brain microvascular endothelial cells revealed that HemoAct significantly suppressed cellular reactive oxygen species production in hypoxia/reoxygenation-treated cells, similar to albumin. Conclusions- HemoAct exerted robust neuroprotection in transient cerebral ischemia. Superior microvascular perfusion with an oxygen delivery capability and possible antioxidant effects appear to be the underlying neuroprotective mechanisms.

Hemoglobin as a Smart pH-Sensitive Nanocarrier To Achieve Aggregation Enhanced Tumor Retention.

Natural proteins have been greatly explored to address unmet medical needs. However, few work has treated proteins as natural pH-sensitive nanoplatforms that make use of the inherent pH gradient of pathogenic sites. Here, hemoglobin is employed as a smart pH-sensitive nanocarrier for near-infrared dye IR780, which disperses well at normal tissue pH and exhibits aggregation at tumor acidic milieu. The pH-sensitive hemoglobin loaded with IR780 shows higher uptake by cancer cells at tumor acidic pH 6.5 than normal tissue pH 7.4. In vivo and ex vivo studies reveal that the hemoglobin nanocarrier exhibits distinct retention kinetics with remarkably prolonged residence time in tumor. Hemoglobin is then proved to be a potent pH-sensitive nanocarrier for cancer diagnosis and treatment.

The effects of hemoglobin glutamer-200 and iNO on pulmonary vascular tone and arterial oxygenation in an experimental acute respiratory distress syndrome.

INTRODUCTION: Hemoglobin-based oxygen carriers (HBOC) have been developed as an alternative to blood transfusions. Their nitric-oxide-scavenging properties HBOC also induce vasoconstriction. In acute lung injury, an excess of nitric oxide results in a general vasodilation, reducing oxygenation by impairing the hypoxic pulmonary vasoconstriction. Inhaled nitric oxide (iNO) is used to correct the ventilation perfusion mismatch. We hypothesized that the additional use of HBOC might increase this effect. In a rodent model of ARDS we evaluated the combined effect of HBOC and iNO on vascular tone and gas exchange. METHODS: ARDS was induced in anaesthetized Wistar rats by saline lavage and aggressive ventilation. Two groups received either hydroxyethylstarch 10% (HES; n = 10) or the HBOC hemoglobin glutamer-200 (HBOC-200; n = 10) via a central venous infusion. Additionally, both groups received iNO. Monitoring of the right ventricular pressure (RVP) and mean arterial pressure (MAP) was performed with microtip transducers. Arterial oxygenation was measured via arterial blood gas analyses. RESULTS: Application of HBOC-200 led to a significant increase of MAP and RVP when compared to baseline and to the HES group. This effect was reversed by iNO. The application of HBOC and iNO had no effect on the arterial oxygenation over time. No difference in arterial oxygenation was found between the groups. CONCLUSION: Application of HBOC led to an increase of systemic and pulmonary vascular resistance in this animal model of ARDS. The increase in RVP was reversed by iNO. Pulmonary vasoconstriction by hemoglobin glutamer-200 in combination with iNO did not improve arterial oxygenation in ARDS.

Sex Difference of Angiotensin IV-, LVV-Hemorphin 7-, and Oxytocin-Induced Antiallodynia at the Spinal Level in Mice With Neuropathic Pain.

BACKGROUND: We demonstrated previously that angiotensin IV (Ang IV) and LVV-hemorphin 7 (LVV-H7) act through the blockade of insulin-regulated aminopeptidase to decrease oxytocin degradation, thereby causing antihyperalgesia at the spinal level in rats. We determined that intrathecal oxytocin can induce significant antihyperalgesia in male rats with inflammation but not in female rats. Thus, we speculate that Ang IV, LVV-H7, and oxytocin can induce antiallodynia, which could be of great therapeutic potential. Because the antihyperalgesia by using these peptides was with sex difference, their possible antiallodynia was examined in male and female mice for comparison. We investigated whether Ang IV, LVV-H7, and oxytocin produce antiallodynia at the spinal level in mice and whether this antiallodynia differs between the sexes. METHODS: Partial sciatic nerve ligation surgery was performed on adult male and female C57BL/6 mice from the same litter (25-30 g). The effects of intrathecal injections of Ang IV (25.8 nmol), LVV-H7 (27.2 nmol), and oxytocin (0.125 or 1.25 nmol) were assessed through the von Frey test 3 days after partial sciatic nerve ligation. RESULTS: Intrathecal injection of Ang IV, LVV-H7, and oxytocin all produced a potent antiallodynia in male mice. However, these antiallodynia effects were either extremely weak or absent in female mice at the same dose. CONCLUSIONS: Intrathecal Ang IV, LVV-H7, and oxytocin can all cause significant antiallodynia in male mice. The Ang IV-, LVV-H7-, and oxytocin-induced antiallodynia effects differed between the sexes at the spinal level in mice.

Assessment of clinical effectiveness of haemoglobin spray as adjunctive therapy in the treatment of sloughy wounds.

OBJECTIVE: To assess use of an adjunctive topical haemoglobin spray in the treatment of sloughy wounds. METHOD: In addition to a standard wound care regimen, consecutive patients with sloughy wounds self-administered haemoglobin spray treatment twice a week until the wound was healed. All patients were followed-up for 26 weeks. Results were compared with a retrospective cohort of 100 consecutive patients, treated during the same period the previous year with standard wound care alone. Data were collected on wound characteristics including percentage of slough, exudate levels, wound pain, and wound size. RESULTS: After 26 weeks, 94/100 patients (94%) treated with haemoglobin spray were completely healed compared with 63/100 control patients (63%). Positive results were evident as early as week one with 52% mean wound size reduction using the heamoglobin spray versus 11% in the retrospective control (p<0.001). At baseline, mean slough coverage was higher in the haemoglobin group, 58% versus 44% in the control group (p<0.001). By week four, mean slough coverage was 1% in the haemoglobin versus 29% in the control group (p<0.001). Reductions in exudate and pain levels (p<0.001) were also observed. CONCLUSION: Overall, results of this evaluation showed the addition of adjunctive haemoglobin spray to standard wound care treatment achieved positive clinical outcomes for patients self-managing complicated sloughy wounds, by supporting reduction of wound exudate and slough within the complex multifaceted process of wound healing.

Effect of Endoplasmic Reticular Stress on Free Hemoglobin Metabolism and Liver Injury.

Elevated soluble (s) CD163 and free hemoglobin (Hb) levels predict fatty liver progression; however, the molecular mechanisms underlying Hb metabolism and liver injury remain undefined. We investigated the effects of endoplasmic reticular (ER) stress on red blood cell (RBC) rheology and free Hb recycling pathways. ER stress was induced in Sprague-Dawley rats by an intraperitoneal injection of tunicamycin (TM) (50, 100, and 200 µg/100 g body weight (BW)) or an intravenous injection of Hb (5 mg/100 g BW). A TM injection increased sCD163 levels, attenuated free Hb uptake, and maintained RBC aggregability. An Hb injection increased serum LVV-hemorphin-7 and total bilirubin levels, but this effect was suppressed by TM. A Western blot analysis showed that ER stress suppressed Hb degradation in the liver through downregulation of globin degradation proteins cathepsin D and glyoxalase-1, as well as heme degradation protein heme oxyganase-1 and keap-1 expression. An ER stress activator also increased the translocation of nuclear factor (NF)-κB (p65) and nuclear factor-erythroid 2-related factor 2 (Nrf2) to nuclei. In conclusion, ER stress triggers ineffective Hb metabolism via altering globin and heme iron degradation pathways. Inability to recycle and metabolize free Hb may underlie the association between iron dysfunction and liver injury.