Alpha-Asarone Ameliorates Neurological Dysfunction of Subarachnoid Hemorrhagic Rats in Both Acute and Recovery Phases via Regulating the CaMKII-Dependent Pathways.

Early brain injury (EBI) is the leading cause of poor prognosis for patients suffering from subarachnoid hemorrhage (SAH), particularly learning and memory deficits in the repair phase. A recent report has involved calcium/calmodulin-dependent protein kinase II (CaMKII) in the pathophysiological process underlying SAH-induced EBI. Alpha-asarone (ASA), a major compound isolated from the Chinese medicinal herb Acorus tatarinowii Schott, was proven to reduce secondary brain injury by decreasing CaMKII over-phosphorylation in rats' model of intracerebral hemorrhage in our previous report. However, the effect of ASA on SAH remains unclear, and the role of CaMKII in both acute and recovery stages of SAH needs further investigation. In this work, we first established a classic SAH rat model by endovascular perforation and intraperitoneally administrated different ASA doses (10, 20, and 40 mg/kg) 2 h after successful modeling. Then, the short- and long-term neurobehavioral performances were blindly evaluated to confirm ASA's efficacy against SAH. Subsequently, we explored ASA's therapeutic mechanism in both acute and recovery stages using histopathological examination, TUNEL staining, flow cytometry, Western-blot, double-immunofluorescence staining, and transmission electron microscopy (TEM) observation. Finally, KN93, a selective CaMKII inhibitor, was applied in oxyhemoglobin-damaged HT22 cells to explore the role of CaMKII in ASA's neuroprotective effect. The results demonstrated that ASA alleviated short- and long-term neurological dysfunction, reduced mortality and seizure rate within 24 h, and prolonged 14-day survival in SAH rats. Histopathological examination showed a reduction of neuronal damage and a restoration of the hippocampal structure after ASA treatment in both acute and recovery phases of SAH. In the acute stage, the Western-blot and flow cytometer analyses showed that ASA restored E/I balance, reduced calcium overload and CaMKII phosphorylation, and inhibited mitochondrion-involved apoptosis, thus preventing neuronal damage and apoptosis underlying EBI post-SAH. In the recovery stage, the TEM observation, double-immunofluorescence staining, and Western-blot analyses indicated that ASA increased the numbers of synapses and enhanced synaptic plasticity in the ipsilateral hippocampi, probably by promoting NR2B/CaMKII interaction and activating subsequent CREB/BDNF/TrkB signaling pathways. Furthermore, KN93 notably reversed ASA's neuroprotective effect on oxyhemoglobin-damaged HT22 cells, confirming CaMKII a potential target for ASA's efficacy against SAH. Our study confirmed for the first time that ASA ameliorated the SAH rats' neurobehavioral deterioration, possibly via modulating CaMKII-involved pathways. These findings provided a promising candidate for the clinical treatment of SAH and shed light on future drug discovery against SAH.

Dexras1 Induces Dysdifferentiation of Oligodendrocytes and Myelin Injury by Inhibiting the cAMP-CREB Pathway after Subarachnoid Hemorrhage.

White matter damage (WMD), one of the research hotspots of subarachnoid hemorrhage (SAH), mainly manifests itself as myelin injury and oligodendrocyte differentiation disorder after SAH, although the specific mechanism remains unclear. Dexamethasone-induced Ras-related protein 1(Dexras1) has been reported to be involved in nervous system damage in autoimmune encephalitis and multiple sclerosis. However, whether Dexras1 participates in dysdifferentiation of oligodendrocytes and myelin injury after SAH has yet to be examined, which is the reason for creating the research content of this article. Here, intracerebroventricular lentiviral administration was used to modulate Dexras1 levels in order to determine its functional influence on neurological injury after SAH. Immunofluorescence, transmission electron microscopy, and Western blotting methods, were used to investigate the effects of Dexras1 on demyelination, glial cell activation, and differentiation of oligodendrocyte progenitor cells (OPCs) after SAH. Primary rat brain neurons were treated with oxyhemoglobin to verify the association between Dexras1 and cAMP-CREB. The results showed that Dexras1 levels were significantly increased upon in vivo SAH model, accompanied by OPC differentiation disturbances and myelin injury. Dexras1 overexpression significantly worsened OPC dysdifferentiation and myelin injury after SAH. In contrast, Dexras1 knockdown ameliorated myelin injury, OPC dysdifferentiation, and glial cell activation. Further research of the underlying mechanism discovered that the cAMP-CREB pathway was inhibited after Dexras1 overexpression in the in vitro model of SAH. This study is the first to confirm that Dexras1 induced oligodendrocyte dysdifferentiation and myelin injury after SAH by inhibiting the cAMP-CREB pathway. This present research may reveal novel therapeutic targets for the amelioration of brain injury and neurological dysfunction after SAH.

Suberoylanilide hydroxamic acid suppresses axonal damage and neurological dysfunction after subarachnoid hemorrhage via the HDAC1/HSP70/TDP-43 axis.

Increased focus has been placed on the role of histone deacetylase inhibitors as crucial players in subarachnoid hemorrhage (SAH) progression. Therefore, this study was designed to expand the understanding of SAH by exploring the downstream mechanism of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in SAH. The expression of TDP-43 in patients with SAH and rat models of SAH was measured. Then, western blot analysis, immunofluorescence staining, and transmission electron microscope were used to investigate the in vitro effect of TDP-43 on a neuronal cell model of SAH established by oxyhemoglobin treatment. Immunofluorescence staining and coimmunoprecipitation assays were conducted to explore the relationship among histone deacetylase 1 (HDAC1), heat shock protein 70 (HSP70), and TDP-43. Furthermore, the in vivo effect of HDAC1 on SAH was investigated in rat models of SAH established by endovascular perforation. High expression of TDP-43 in the cerebrospinal fluid of patients with SAH and brain tissues of rat models of SAH was observed, and TDP-43 accumulation in the cytoplasm and the formation of inclusion bodies were responsible for axonal damage, abnormal nuclear membrane morphology, and apoptosis in neurons. TDP-43 degradation was promoted by the HDAC1 inhibitor SAHA via the acetylation of HSP70, alleviating SAH, and this effect was verified in vivo in rat models. In conclusion, SAHA relieved axonal damage and neurological dysfunction after SAH via the HSP70 acetylation-induced degradation of TDP-43, highlighting a novel therapeutic target for SAH.

Polymeric nanoparticles for hemoglobin-based oxygen carriers.

This article reports on the current status of the research on blood substitutes with particular attention on hemoglobin-based oxygen carriers (HBOCs). Insights on the physiological role of hemoglobin are reported in the view of the development of both acellular and cellular hemoglobin-based oxygen carriers. Attention is then focused on biocompatible polymeric materials that find application as matrices for cellular based HBOCs and on the strategies employed to avoid methemoglobin formation. Results are reported regarding the use of bioerodible polymeric matrices based on hemiesters of alternating copolymer (maleic anhydride-co-butyl vinyl ether) for the preparation of hemoglobin loaded nanoparticles.

Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC).

Acellular hemoglobins developed as oxygen bridging agents with volume expanding properties ("blood substitutes") are prone to autoxidation and oxidant-mediated structural changes in circulation. In the presence of hydrogen peroxide and either ascorbate or urate we show that ferric hemoglobin functions as a true enzymatic peroxidase. The activity saturates with both substrates and is linearly dependent on protein concentration. The activity is enhanced at low pH with a pKa of 4.7, consistent with protonation of the ferryl species (Fe(IV)-OH) as the active intermediate. To test whether these redox reactions define its behaviour in vivo we exchanged transfused guinea pigs with 50% polymerized bovine Hb (PolyHbBv) and monitored plasma levels of endogenous ascorbate and urate. Immediately after transfusion, met PolyHbBv levels increased up to 30% of total Hb and remained at this level during the first 24 h post transfusion. Plasma ascorbate decreased by 50% whereas urate levels remained unchanged after transfusion. A simple kinetic model, assuming that ascorbate was a more active ferric heme reductase and peroxidase substrate than urate, was consistent with the in vivo data. The present finding confirms the primary and secondary roles of ascorbate and urate respectively in maintaining the oxidative stability of infused Hb.

Ligand reactivity and allosteric regulation of hemoglobin-based oxygen carriers.

Historically, exogenous administration of hemoglobin solutions to implement the oxygen transport capacity for clinical applications suffered from dramatic drawbacks, resulting in the failure of many attempts. In the last decades, the biochemical and physiological basis responsible for the therapeutic failures has been extensively investigated. It is now widely accepted that they mostly arise because, out of the confined and controlled environment of the red blood cell, hemoglobin exhibits tetramer instability, increased auto-oxidation rate, higher oxygen affinity, altered cooperativity and nitric oxide reactivity. Moreover, it became evident that the design of a hemoglobin-based oxygen carrier that exactly reproduces the "physiological" oxygen-binding curve is not only an overly ambitious task, but may also represent a wrong approach for many potential clinical applications. Under these premises, and given the complex chemical nature of blood, it is obvious that any strategy undertaken to modify the stability and function of the hemoglobin tetramer for clinical use should be driven by a detailed knowledge of its structure, dynamics and mechanism of allosteric regulation. We briefly review the most recent theories and experiments that increased our understanding of the mechanism of homo- and heterotropic effects in human hemoglobin, trying to interpret, on a biophysical basis, how diverse approaches like polymerization, cross-linking, site-directed mutagenesis, surface decoration and encapsulation may affect ligand affinity and allosteric regulation.

Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration.

Blood transfusion is still a critical therapy in many diseases, traumatic events and war battlefields. However, blood cross-matching and storage may limit its applicability, especially in Third World countries. Moreover, haemoglobin, which in red blood cells is the key player in the oxygen transport from lung to tissues, when free in the plasma causes hypertension and renal failure. This investigation was aimed at the development of a novel haemoglobin-based oxygen carrier with low vasoactivity and renal filtration properties. Human haemoglobin was chemically conjugated with polyethylene glycol (PEG) under either aerobic or anaerobic conditions, following different chemical procedures. The resulting PEGylated haemoglobin products were characterized in terms of oxygen affinity, cooperativity, effects of protons and carbon dioxide concentration, and oxidation stability, and were transfused into rats to evaluate vasoactivity and renal filtration. A deoxyhaemoglobin, conjugated with seven PEG and seven propionyl groups, which we called Euro-PEG-Hb, did not produce profound hypertension, was 99% retained within 6 h, and exhibited oxygen binding properties and allosteric effects more similar to human haemoglobin A than the other tested PEGylated haemoglobin derivatives, thus appearing a very promising candidate as blood substitute.

Hb(alphaalpha,betabeta): a novel fusion construct for a dimeric, four-domain hemoglobin.

Hemoglobin-based blood substitutes are one of the options available to derive a resuscitating fluid taking into account clinical and physiological demands. In this paper we investigated a novel protein, Hb(alphaalpha,betabeta) obtained as a combination of two homodimers alpha(2) and beta(2) both derived from a fusion gene containing two alfa chains or two beta chains, each respectively coupled via a specific linker. The construct here described is thus a novel heterodimeric hemoglobin carrying four heme groups. The protein cannot dissociate into dimers, as demonstrated by its absence of reactivity versus haptoglobin, and is expected to have a relatively long circulating half-life. The modification does not increase the autoxidation rate, but increases the oxygen affinity, due to a destabilization of the T quaternary state. Characterization of the biochemical properties of this protein in comparison with HbA is reported.

Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers.

The cerebrovascular effects of exchange transfusion of various cell-free hemoglobins that possess different oxygen affinities are reviewed. Reducing hematocrit by transfusion of a non-oxygen-carrying solution dilates pial arterioles on the brain surface and increases cerebral blood flow to maintain a constant bulk oxygen transport to the brain. In contrast, transfusion of hemoglobins with P50 of 4-34 Torr causes constriction of pial arterioles that offsets the decrease in blood viscosity to maintain cerebral blood flow and oxygen transport. The autoregulatory constriction is dependent on synthesis of 20-HETE from arachidonic acid. This oxygen-dependent reaction is apparently enhanced by facilitated oxygen diffusion from the red cell to the endothelium arising from increased plasma oxygen solubility in the presence of low or high-affinity hemoglobin. Exchange transfusion of recombinant hemoglobin polymers with P50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P50 as high as red blood cell hemoglobin to facilitate oxygen delivery.

All hemoglobin-based oxygen carriers are not created equally.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) also known as "blood substitutes" have been under active clinical development over the last two decades. Cell-free Hb outside its natural protective red blood cell environment, as is the case with all HBOCs, has been shown to be vasoactive in part due to the scavenging of vascular endothelial nitric oxide (NO) and may in some instances induce heme-mediated oxidative stress. Chemical modification intended to stabilize HBOCs in the tetrameric or polymeric forms introduces conformational constraints that result in proteins with diverse allosteric responses as well as oxidative and nitrosative redox side reactions. Intra and inter-molecular cross-linking may in some instances also determine the interactions between HBOCs and normal oxidative inactivation and clearance mechanisms. Oxygen and oxidative reactions of normal and several cross-linked Hbs as well as their interactions with endogenous plasma protein (haptoglobin) and cellular receptor pathways (macrophage CD163) differ significantly. Therefore, safety and efficacy may be addressed by designing HBOCs with modifications that limit hypertension, minimize heme destabilization and take into account endogenous Hb removal mechanisms to optimize exposure times for a given indication.

Blood on tap. Part 1. History in the making.

Emergency services around the United States are about to become part of the front lines in the race to bring to market the first blood substitute with oxygen-carrying capabilities. Take a look inside the high-tech world of biopharmaceuticals and the innovative pioneers who are chasing after a scientific holy grail; a synthetic substitute for blood. In the process, they have made and lost fortunes, advanced our understanding of the nature of blood and launched one of the biggest ethical controversies in modern medical history. As phase three clinical trials move to the prehospital arena, biotechnology firms are staking everything on the notion that they're about to change the way we treat trauma patients. They may be right.

Heart protection by cardioplegic solutions containing oxyhemoglobin pretreated by carbontetrachloride and freeze-drying with sucrose.

A technologically improved variant of native stroma-free oxyhemoglobin (SFH) pretreated by carbontetrachloride and freeze-drying with 240 mM sucrose were reconstituted in a properly diluted ionic solution to reach the final concentration of 66 g oxyhemoglobin/L, osmolality 280-320 m0sm and pH 7.4. Cardioplegia of isolated rat heart was induced and maintained by this solution without recirculation for 3 h at 20 degrees C prior to heterotopic allo-transplantation of the graft. Evaluation of the survival and performance of each graft after 24 h and extent of tissue necroses indicated that the given standardly produced SFH variant ensured reproducible heart preservation from ischemic and reperfusion injury similarly as did the renown crystalloid cardioplegic solution CUSTODIOL.

A potential blood substitute from carboxylic dextran and oxyhemoglobin. II. Physicochemical and physiological assessments. Preliminary results on guinea pig.

The hemoglobin solution covalently linked to dextran benzene tetracarboxylate (dex-BTC) described in Part I, offered the following characteristics: [Hb] = 70 g/l, non modified Hb less than 15%, metHb less than 5%, P50 close to blood value, viscosity and oncotic pressure near to physiological values (37 degrees C). It was biocompatible, stable in plasma, non hypotensive in rat model, non pyrogenic and did not present abnormal toxicity (on mice, according to the french Pharmacopea). Nevertheless, on account of the presence of dextran in the conjugate and due to the literature, we observed an anaphylactoïd reaction in rats consecutive to the injections: absence of hypotension but presence of a massive oedema resulting from an important increase in the capillary permeability with harmful consequences to the vascular retention and survival time. As guinea pigs are insensitive to dextran, we are beginning to assess this conjugate solution in this animal. The first results are very promising: a lack of mortality after large injection, an almost complete vascular retention, a weak urinary loss (only non conjugated Hb), and a plasmatic half-disappearance time close to 7h. Results from haemorrhagic shocks to a final hematocrit less than 0.10 l/l and total and isovolemic exchange transfusions seem to prove a real oxygen-carrying capacity of this new Hb solution.