Hemoglobin Extravasation Research Articles

Optimization and Investigation of Hemoglobin‐Loaded ZIF‐90 Metal–Organic Framework Nanoparticles as Artificial Oxygen Carriers

AbstractBlood transfusions are vital, yet limited shelf‐life and storage conditions of red blood cells (RBCs) hinder their use in emergencies. Hemoglobin‐based oxygen carriers (HBOCs) aim to address these challenges, but previous versions faced clinical setbacks due to safety concerns related to hemoglobin (Hb) extravasation. ZIF‐90, a novel metal–organic framework variant where imidazole‐2‐carboxaldehyde (2‐ICA) is bridged to Zn2+ ions, is explored. Optimizing Zn2+:2‐ICA ratios and Hb concentrations, Hb‐loaded ZIF‐90 nanoparticles (NPs) are synthesized in one‐step and under mild synthesis conditions. These NPs achieve a 25.1% yield, 7.0 mg mL−1 Hb concentration, 70.3% encapsulation efficiency (EE), and 96.4% drug loading. They exhibit a left‐shifted oxygen dissociation curve with p50 of ≈8 mmHg, indicating enhanced oxygen release at lower partial oxygen pressures as compared to native RBCs. This feature makes Hb‐loaded ZIF‐90 NPs suitable for certain medical applications including ischemia‐reperfusion injury management. Furthermore, the impact of two prominent capping agents: polyvinylpyrrolidone and polyethylene glycol (PEG) is assessed. PEG improves 2‐ICA incorporation and stabilizes the ZIF‐90 crystalline phase, albeit with reduced yield, Hb content, and EE. The findings underscore the potential of Hb‐loaded ZIF‐90 NPs as next‐generation HBOCs, offering enhanced uniformity, high Hb content, and efficient oxygen binding and release properties for medical applications.

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham. Here we develop a method for measuring Hb extravasation in brain. We exchange transfused rats by haemorrhaging 50% of their blood with simultaneous, isovolemic replacement with Hextend (negative control), MP4OX, or αα-cross-linked Hb. Animals were sacrificed 2 h after transfusion, brain tissue was harvested and processed for double-staining immunofluorescence, whereby Hb ? chain and NeuN (a neuron protein) were stained and quantitated. Whereas Hextend did not induce Hb extravasation, in both MP4OX and ??Hb brains Hb molecules were detected outside neurons. The level of extravasated Hb chains was > 3-fold higher in Hb compared to MP4OX. Western blot analysis revealed that the expression levels of protein related to redox imbalance (e.g., Nrf2, iNOS and ERK phosphorylation) were higher in ααHb than MP4OX. In conclusions, higher Hb extravasation in ααHb than MP4OX induces redox imbalance, which causes higher anti-oxidant response. Whereas Nrf2 response may be considered protective, iNOS response appears damaging.

Modeling hemoglobin and hemoglobin:haptoglobin complex clearance in a non-rodent species–pharmacokinetic and therapeutic implications

Background: Haptoglobin (Hp) prevents hemoglobin (Hb) extravasation and attenuates Hb induced tissue oxidation and vasoconstriction. Small animal models such as mouse, rat and guinea pig appear to demonstrate proof-of-concept for Hb neutralization by Hp in diverse pre-clinical conditions. However, these species differ significantly from humans in the clearance of Hb:Hp and demonstrate long persistence of circulating Hb:Hp complexes.Objective: The focus of this study is to understand Hb:Hp clearance in a non-rodent species. In contrast to rodents, dogs maintain high plasma Hp concentrations comparable to humans and demonstrate more rapid clearance of Hb:Hp when compared to rodent species, therefore dogs may represent a relevant species to evaluate Hb:Hp pharmacokinetics and cellular clearance.Results: In this study we show, that like human macrophages, dog peripheral blood monocyte derived macrophages express a glucocorticoid inducible endocytic clearance pathways with a high specificity for the Hb:Hp complex. Evaluating the Beagle dog as a non-rodent model species we provide the first pharmacokinetic parameter estimates of free Hb and Hb:Hp complexes. The data demonstrate a significantly reduced volume of distribution (Vc) for Hb:Hp compared to free Hb, increased maximum plasma concentrations and areas under plasma concentration time curves (Cmax and AUC). Significantly reduced total body clearance (CL) and a longer terminal half-life (t1/2) of approximately 12 h were also observed for the Hb:Hp complex. Distribution and clearance were identical for dimeric and multimeric Hb:Hp complexes. We found no significant effect of a high-dose glucocorticoid treatment protocol on Hb:Hp pharmacokinetic parameter estimates.Conclusion: Collectively, our study supports the dog as a non-rodent animal model to study pharmacological and pharmacokinetic aspects of Hb clearance systems and apply the model to studying Hp as a therapeutic in diseases of hemolysis.

Combination treatment of r-tPA and an optimized human apyrase reduces mortality rate and hemorrhagic transformation 6 h after ischemic stroke in aged female rats

Recombinant tissue plasminogen activator (r-tPA) is the only FDA-approved drug treatment for ischemic stroke and must be used within 4.5h. Thrombolytic treatment with r-tPA has deleterious effects on the neurovascular unit that substantially increases the risk of intracerebral hemorrhage if administered too late. These therapeutic shortcomings necessitate additional investigation into agents that can extend the therapeutic window for safe use of thrombolytics. In this study, combination of r-tPA and APT102, a novel form of human apyrase/ADPase, was investigated in a clinically-relevant aged-female rat embolic ischemic stroke model. We propose that successfully extending the therapeutic window of r-tPA administration would represent a significant advance in the treatment of ischemic stroke due to a significant increase in the number of patients eligible for treatment. Results of our study showed significantly reduced mortality from 47% with r-tPA alone to 16% with co-administration of APT102 and r-tPA. Co-administration decreased cortical (47±5% vs. 29±5%), striatal (50±2%, vs. 40±3%) and total (48±3%vs. 33±4%) hemispheric infarct volume compared to r-tPA alone. APT102 improved neurological outcome (8.9±0.6, vs. 6.8±0.8) and decreased hemoglobin extravasation in cortical tissue (1.9±0.1mg/dl vs. 1.4±0.1mg/dl) striatal tissue (2.1±0.3mg/dl vs. 1.4±0.1mg/dl) and whole brain tissue (2.0±0.2mg/dl vs. 1.4±0.1mg/dl). These data suggest that APT102 can safely extend the therapeutic window for r-tPA mediated reperfusion to 6h following experimental stroke without increased hemorrhagic transformation. APT102 offers to be a viable adjunct therapeutic option to increase the number of clinical patients eligible for thrombolytic treatment after ischemic stroke.

Hemoglobin as a source of iron overload in multiple sclerosis: does multiple sclerosis share risk factors with vascular disorders?

Although iron is known to be essential for the normal development and health of the central nervous system, abnormal iron deposits are found in and around multiple sclerosis (MS) lesions that themselves are closely associated with the cerebral vasculature. However, the origin of this excess iron is unknown, and it is not clear whether this is one of the primary causative events in the pathogenesis of MS, or simply another consequence of the long-lasting inflammatory conditions. Here, applying a systems biology approach, we propose an additional way for understanding the neurodegenerative component of the disease caused by chronic subclinical extravasation of hemoglobin, in combination with multiple other factors including, but not limited to, dysfunction of different cellular protective mechanisms against extracellular hemoglobin reactivity and oxidative stress. Moreover, such considerations could also shed light on and explain the higher susceptibility of MS patients to a wide range of cardiovascular disorders.

Neuregulin-1 Effects on Endothelial and Blood–Brain Barrier Permeability After Experimental Injury

Blood-brain-barrier disruption occurs with a high incidence after traumatic brain injury, and is an important contributor to many pathological processes, including brain edema, inflammation, and neuronal cell death. Therefore, blood-brain-barrier integrity is an important potential therapeutic target in the treatment of the acute phase of brain trauma. In this short communication, we report our data showing that neuregulin-1 (NRG1), a growth factor with diverse functions in the CNS, ameliorates pathological increases in endothelial permeability and in BBB permeability in experimental models of injury. For in-vitro studies, rat brain endothelial cells were incubated with the inflammatory cytokine IL-1β, which caused an increase in permeability of the cell layer. Co-incubation with NRG1 ameliorated this permeability increase. For in-vivo studies, C57Bl mice were subjected to controlled cortical impact (CCI) under anesthesia, and BBB permeability was assessed by measuring the amount of Evans blue dye extravasation at 2h. NRG1 administered by tail-vein injection 10 minutes after CCI resulted in a decrease in Evans blue dye extravasation by 35%. Since Evans blue extravasation may result from an increase in BBB permeability or from bleeding due to trauma, hemoglobin ELISA was also performed at the same time point. There was a trend towards lower levels of hemoglobin extravasation in the NRG1 group, but the results did not reach statistical significance. MMP-9 activity was not different between groups at 2h. These data suggest that NRG1 has beneficial effects on endothelial permeability and BBB permeability following experimental trauma, and may have neuroprotective potential during CNS injury.

Oxygen Therapy Reduces Secondary Hemorrhage after Thrombolysis in Thromboembolic Cerebral Ischemia

Hyperbaric oxygen (HBO) and normobaric hyperoxia (NBO) protect the brain parenchyma and the cerebral microcirculation against ischemia. We studied their effect on secondary hemorrhage after thrombolysis in two thromboembolic middle cerebral artery occlusion (MCAO) (tMCAO) models. Beginning 60 minutes after tMCAO with either thrombin-induced thromboemboli (TT) or calcium-induced thromboemboli (CT), spontaneously hypertensive rats (n=96) breathed either air, 100% O(2) (NBO), or 100% O(2) at 3 bar (HBO) for 1 hour. Immediately thereafter, recombinant tissue plasminogen activator (rt-PA, 9 mg/kg) was injected. Although significant reperfusion was observed after thrombolysis in TT-tMCAO, vascular occlusion persisted in CT-tMCAO. In TT-tMCAO, NBO and HBO significantly reduced diffusion-weighted imaging-magnetic resonance imaging (MRI) lesion volume and postischemic blood-brain barrier (BBB) permeability on postcontrast T1-weighted images. NBO and, significantly more potently, HBO reduced macroscopic hemorrhage on T2* MRI and on corresponding postmortem cryosections. Oxygen therapy lowered hemoglobin content and attenuated activation of matrix metalloproteinases in the ischemic hemisphere. In contrast, NBO and HBO failed to reduce infarct size in CT but both decreased BBB damage and microscopic hemorrhagic transformation. Only HBO reduced hemoglobin extravasation in the ischemic hemisphere. In conclusion, NBO and HBO decrease infarct size after thromboembolic ischemia only if recanalization is successful. As NBO and HBO also reduce postthrombolytic intracerebral hemorrhage, combining the two with thrombolysis seems promising.

Hemoglobin-based Oxygen Carriers

Hemoglobin-based Oxygen Carriers

Protection of cerebral microvasculature after moderate hypothermia following experimental focal cerebral ischemia in mice

Clinical studies have shown that the treatment of ischemic stroke with hypothermia is promising. In this animal study, we investigated the fate of the microvasculature following focal cerebral ischemia in mice with and without hypothermia. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery (MCAO) (3 h) with an intraluminal filament technique. Eight mice received normothermia (36.5 °C, NT) and eight received hypothermia (32–34 °C, HT) treatment during 24 h of reperfusion. Another six mice represented the sham group. Analysis of the hypothermic group in comparison to the normothermic group revealed a significantly reduced infarct volume (NT: 63.56 ± 4.62 mm 3 SEM, HT: 38.09 ± 4.83 mm 3 SEM; P < 0.01) and showed considerably ameliorated neurological deficits (Garcia-score) after 24 h ( P < 0.01). In addition, the degradation of the microvascular basal lamina antigen collagen type IV after normothermia was strongly reduced ( P < 0.05) compared to sham. Hypothermia diminished this effect so that collagen type IV was not significantly reduced compared to sham. Moreover the hemoglobin extravasation was strongly reduced under hypothermic treatment compared to the normothermic group ( P < 0.01). In the hypothermia group the urokinase plasminogen-activator (uPA) activity ( P = 0.01) was significantly decreased compared to the normothermia group. Also MMP-9 was significantly reduced ( P < 0.05) during hypothermic treatment. In conclusion, for the first time we show in mice that hypothermia preserves the microvascular wall structures after ischemia. We have demonstrated that hypothermia protects the basal lamina, reduces the infarct volume and hemorrhage, and reduces proteolytic enzymes. These protective effects in an additional animal model of ischemia and reperfusion strongly recommend hypothermia as a potential beneficial treatment for stroke.

Rt-PA causes a dose-dependent increase in the extravasation of cellular and non-cellular blood elements after focal cerebral ischemia

While recombinant tissue plasminogen activator (rt-PA) is successfully used for thrombolysis in human stroke, it may increase the risk of hemorrhagic complications. We describe the effects of different doses of rt-PA (saline, 0.9, 9, or 18 mg rt-PA/kg body weight) on the extravasation of blood components following experimental cerebral ischemia (3 h, 24 h reperfusion, suture model) in rats. The damage to the blood–brain barrier and the hemoglobin extravasation were quantified by Western blotting and immunohistochemistry. Both were significantly elevated in the ischemic cortex and basal ganglia. As rt-PA doses rose, the hemoglobin content as well as the damage to the blood–brain barrier in the ischemic side also rose significantly ( p < 0.001). This correlated significantly with the rising MMP-9 (matrix metalloproteinase) after increasing doses of rt-PA. Despite various benefits, rt-PA is responsible for a dose-dependent increase of edema and hemorrhage after cerebral ischemia. Clinicians should consider using the lowest effective dose of rt-PA in stroke patients.

Liposome-Encapsulated Hemoglobin Reduces the Size of Cerebral Infarction in the Rat

Liposome-encapsulated hemoglobin (LEH; TRM-645) is a novel oxygen (O(2)) carrier with a lower O(2) affinity (P(50)O(2)=40 mm Hg) than red blood cells. In contrast to cell-free hemoglobin, encapsulation prevents hemoglobin extravasation, whereas its subcellular size (230 nm) may improve O(2) delivery and limit the severity of cerebral infarction. The extent of cerebral infarction was determined 24 hours after photochemically induced thrombosis of the middle cerebral artery from the integrated area of infarction detected by triphenyltetrazolium chloride staining in rats receiving no treatment, 10 mL/kg of LEH, homologous blood, empty liposomes, or saline. To develop a dose-response relationship, LEH dose was reduced from 10 mL/kg to 2 mL/kg, 0.4 mL/kg, and 0.08 mL/kg. Infarction extent was significantly suppressed in rats receiving LEH as compared with animals receiving no infusion, saline, empty liposome, or transfusion in the cortex but not in the basal ganglia, where all had similar degrees of damage. The dose-response relationship revealed that as little as 2 mL/kg of LEH was protective, whereas the total blood O(2) content, hemoglobin level, and transfusion and/or infusion of empty liposomes or saline were not effective. Our results suggest that LEH significantly reduces the area of infarction in the cortex but not in basal ganglia after photochemically induced thrombosis of the middle cerebral artery in the rat.

Development of Zero-Link Polymers of Hemoglobin, Which do not Extravasate and do not Induce Pressure Increases upon Infusion

Intramolecular crosslink of hemoglobin tetramers solved the problem of urine elimination and short intravascular retention time of cell free hemoglobin infusion. It also produced a family of crosslinked hemoglobins with P50 between 18 and 30 mmHg. However, it did not solve the problem of MAP increases in infused animals. It was proven that extravasation of hemoglobin into interstitial fluid was responsible for MAP increases. Extravasation and the MAP increase was avoided using a hemoglobin polymer with average size near 25 MDa. In spite of a very high oxygen affinity, this polymer delivered oxygen to tissues, producing either vasodilation or vasoconstriction according to oxygen needs. It was also proven that cell free hemoglobins are more efficient than red cells in delivering oxygen to tissues.

Doxycycline inhibits MMPs via modulation of plasminogen activators in focal cerebral ischemia

Tetracyclines inhibit matrix metalloproteinases (MMPs) and reduce infarction volume following cerebral ischemia. In this thesis an involvement of urokinase could be proven. Cerebral ischemia in rats was induced for 3 h followed by 24 h reperfusion (suture model). Each 6 animals received orally either doxycycline or water. Doxycycline treatment began 10 days before ischemia. MMP-2 and MMP-9 were substantially decreased. The possibility of involvement of the endogenous MMP inhibitors in the MMP inhibiting mechanisms was excluded. The plasminogen activator uPA was significantly decreased by doxycycline indicating an MMP inhibiting mechanism including the plasminogen/plasmin system. In the doxycycline group, this resulted in a decreased damage to the cerebral microvessels and less loss of the basal lamina antigen collagen type IV. Hemoglobin extravasation was also significantly reduced. Our results suggest that doxycycline may have a potential use as an anti-ischemic compound since it provides microvascular protection by inhibiting the plasminogen system.

Computation of plasma hemoglobin nitric oxide scavenging in hemolytic anemias

Intravascular hemoglobin limits the amount of endothelial-derived nitric oxide (NO) available for vasodilation. Cell-free hemoglobin scavenges NO more efficiently than red blood cell-encapsulated hemoglobin. Hemolysis has recently been suggested to contribute to endothelial dysfunction based on a mechanism of NO scavenging by cell-free hemoglobin. Although experimental evidence for this phenomenon has been presented, support from a theoretical approach has, until now, been missing. Indeed, due to the low amounts of cell-free hemoglobin present in these pathological conditions, the role of cell-free hemoglobin scavenging of NO in disease has been questioned. In this study, we model the effects of cell-free hemoglobin on NO bioavailability, focusing on conditions that closely mimic those under known pathological conditions. We find that as little as 1 μM cell-free intraluminal hemoglobin (heme concentration) can significantly reduce NO bioavailability. In addition, extravasation of hemoglobin out of the lumen has an even greater effect. We also find that low hematocrit associated with anemia increases NO bioavailability but also leads to increased susceptibility to NO scavenging by cell-free hemoglobin. These results support the paradigm that cell-free hemoglobin released into plasma during intravascular hemolysis in human disease contributes to the experimentally observed reduction in NO bioavailability and endothelial dysfunction.

Pravastatin reduces microvascular basal lamina damage following focal cerebral ischemia and reperfusion

Transient ischemia has been shown to damage the basal lamina of the cerebral microvasculature. Other studies proved statins to be beneficial to non-cerebral microvessels. The aim of this study was to determine whether pravastatin pretreatment ameliorates microvascular basal lamina damage following transient ischemia. Using the suture model, we subjected 15 rats to focal ischemia (3 h) and reperfusion (24 h). Rats received pravastatin (20 mg/kg/day) or saline for 4 weeks prior to the experiment. The outcome was determined by a behavior test and the infarct size. Collagen type IV, a marker for an intact basal lamina, and hemoglobin extravasation were measured by Western blot analysis. A ratio (in percentage) between ischemic and contralateral hemispheres was calculated. Pravastatin pretreatment resulted in a significantly better neurological outcome and reduced infarct size (15 +/- 0.5 and 59 +/- 10 mm(3), respectively) compared with controls (12.25 +/- 0.4 and 167 +/- 13 mm(3), respectively, P < 0.01 for both). In controls, loss of collagen type IV was seen in the basal ganglia and in the cortex (43 +/- 4 and 64 +/- 5%, respectively). Pravastatin prevented significant collagen loss (basal ganglia: 106 +/- 17%; cortex: 112 +/- 14%, P < 0.01 for both) and significantly reduced the hemoglobin extravasation compared with controls in the basal ganglia (198 +/- 49 vs. 553 +/- 47%, P < 0.01). Pravastatin pretreatment resulted in a reduction of microvascular basal lamina damage and hemoglobin extravasation following transient ischemia. Pravastatin seems to protect the cerebral microvascular system.

Keeping cool in acute liver failure: Rationale for the use of mild hypothermia

Encephalopathy, brain edema and intracranial hypertension are neurological complications responsible for substantial morbidity/mortality in patients with acute liver failure (ALF), where, aside from liver transplantation, there is currently a paucity of effective therapies. Mirroring its cerebro-protective effects in other clinical conditions, the induction of mild hypothermia may provide a potential therapeutic approach to the management of ALF. A solid mechanistic rationale for the use of mild hypothermia is provided by clinical and experimental studies showing its beneficial effects in relation to many of the key factors that determine the development of brain edema and intracranial hypertension in ALF, namely the delivery of ammonia to the brain, the disturbances of brain organic osmolytes and brain extracellular amino acids, cerebro-vascular haemodynamics, brain glucose metabolism, inflammation, subclinical seizure activity and alterations of gene expression. Initial uncontrolled clinical studies of mild hypothermia in patients with ALF suggest that it is an effective, feasible and safe approach. Randomized controlled clinical trials are now needed to adequately assess its efficacy, safety, clinical impact on global outcomes and to provide the guidelines for its use in ALF.

Regional myocardial perfusion under exchange transfusion with liposomal hemoglobin: in vivo and in vitro studies using rat hearts

The purpose of this study was to test the hypothesis that exchange transfusion with liposomal hemoglobin (LH) reduces the microheterogeneity of regional myocardial flows while sustaining cardiac function. Neo Red Cell mixed with albumin was used as the LH solution, in which the LH volume fraction was 17 approximately 18% and hemoglobin density was nearly two-thirds smaller than in rat blood. Regional myocardial flows in left ventricular free walls were measured by tracer digitalradiography (100-mum resolution) in anesthetized rats with or without 50% blood-LH exchange transfusion. Within-layer flow distributions showed lower heterogeneity with (n = 8) than without (n = 8) LH transfusion. No extravasation of hemoglobin was confirmed by 3,3-diaminobenzidin staining (n = 2). Carotid flow increased by 68% due to LH transfusion, whereas arterial pressure and heart rate remained unchanged. On the other hand, cross-circulated rat hearts (n = 7) were used to evaluate the effects of 50% blood-LH exchange on coronary flow and tone preservation under 300-beats/min pacing and 100-mmHg perfusion pressure. Blood-LH exchange caused a 71% increase of coronary flow and 10% decrease of percent flow increase during hyperemia after 30-s flow interruption. Myocardial O(2) supply and consumption increased by 9% and 10%, respectively, whereas myocardial O(2) extraction remained unchanged. The large increases of in vivo carotid flow and coronary flow in cross-circulated hearts due to LH coperfusion could be explained by the reduction of apparent flow viscosity. These results suggest that under LH coperfusion, the microheterogeneity of myocardial flows decreases with increased coronary flow while fairly preserving coronary tone and cardiac function.

Starch-hemoglobin Induces Contraction on Isolated Rat Aortic Rings

Blood substitutes are being developed using molecular solutions of modified free hemoglobin; however, anaphylactic reactions, severe renal toxicity, and hypertension have been reported in experimental models and human beings. Hypertension remains as an obstacle to the clinical use of most blood substitutes. Several investigators suggest that this effect is due to the interaction between nitric oxide and hemoglobin into the endothelial cells; hence, prevention of hemoglobin extravasation would avoid vasoconstriction. The forms of hemoglobin likely to prevent extravasation include polymerized and encapsulated Hb. Another alternative and significantly less expensive approach is the hydroxyethyl starch Hb-polymer. The aim of the present study was to compare the effect of hydroxyethyl-starch-hemoglobin with that of stroma-free hemoglobin on the in vitro contractile activity of aortic rings isolated from adult male rats. The hemoglobin-based oxygen carrier was made using stroma-free hemoglobin prepared from outdated human red cells and conjugated with 10% hydroxyethyl starch 200-260 MW. The experiments were made in thoracic segments of the aortic rings incubated with hemoglobin, starch-hemoglobin or Ringer Krebs-Bicarbonate solution (RKB) during 30 min. Smooth muscle contraction with phenylephrine and subsequent inhibition of contraction with carbachol were performed before and after incubation with hemoglobin, starch-hemoglobin, or vehicle. Incubation with hemoglobin and starch-hemoglobin significantly increased the contractile response to phenylephrine of aortic rings compared with RKB solution. The maximal response to carbachol was significantly decreased in the aortic rings incubated with either hemoglobin or starch-hemoglobin in comparison with the RKB-incubated tissues. There were no differences between the aortic rings incubated with either hemoglobin, or starch-hemoglobin. These results show that there are no differences between the effects of stroma-free hemoglobin and starch-hemoglobin on the in vitro contractile activity of aortic rings isolated from adult male rats. Our findings do not support the hypothesis that an increase in the size of the hemoglobin molecule prevents hemoglobin extravasation, and the consequent vasoconstriction due to the scavenging of nitric oxide by stroma free hemoglobin in the cellular space between endothelium and smooth muscle.

The effect of brain temperature on hemoglobin extravasation after traumatic brain injury

Although the benefits of posttraumatic hypothermia have been reported in experimental studies, the potential for therapeutic hypothermia to increase intracerebral hemorrhage remains a clinical concern. The purpose of this study was to quantify the amount of extravasated hemoglobin after traumatic brain injury (TBI) and to assess the changes in intracerebral hemoglobin concentrations under posttraumatic hypothermic and hyperthermic conditions. Intubated and anesthetized rats were subjected to fluid-percussion injury (FPI). In the first experiment, rats were divided into moderate (1.8-2.2 atm) and severe (2.4-2.7 atm) TBI groups. In the second experiment, the effects of 3 hours of posttraumatic hypothermia (33 or 30 degrees C), hyperthermia (39 degrees C), or normothermia (37 degrees C) on hemoglobin levels following moderate trauma were assessed. The rats were perfused with saline at 24 hours postinjury, and then the traumatized and contralateral hemispheres, including the cerebellum, were dissected from whole brain. The hemoglobin level in each brain was quantified using a spectrophotometric hemoglobin assay. The results of these assays indicate that moderate and severe FPI induce increased levels of hemoglobin in the ipsilateral hemisphere (p < 0.0001). After severe TBI, the hemoglobin concentration was also significantly increased in the contralateral hemisphere (p < 0.05) and cerebellum (p < 0.005). Posttraumatic hypothermia (30 degrees C) attenuated hemoglobin levels (p < 0.005) in the ipsilateral hemisphere, whereas hyperthermia had a marked adverse effect on the hemoglobin concentration in the contralateral hemisphere (p < 0.05) and cerebellum (p < 0.005). Injury severity is an important determinant of the degree of hemoglobin extravasation after TBI. Posttraumatic hypothermia reduced hemoglobin extravasation, whereas hyperthermia increased hemoglobin levels compared with normothermia. These findings are consistent with previous data reporting that posttraumatic temperature manipulations alter the cerebrovascular and inflammatory consequences of TBI.

Vascular response to infusions of a nonextravasating hemoglobin polymer.

The clinical utility of cross-linked tetrameric hemoglobin solutions is limited by peripheral vasoconstriction thought to be due to scavenging of nitric oxide. In addition, transfusion of crude preparations of hemoglobin polymers can cause arterial hypertension. We tested the hypothesis that eliminating low-molecular-weight components from the polymer solution would prevent extravasation and its associated pressor response. A zero-link polymer of bovine hemoglobin was developed without chemical linkers left between the tetramers. Transfusion of unprocessed preparations of these polymers in rats resulted in appearance of the polymer in the renal hilar lymph. However, eliminating the low-molecular-weight components with a 300-kDa diafiltration resulted in an average hydrodynamic radius of 250 A and in undetectable levels of polymer in hilar lymph. Exchange transfusion in anesthetized rats and cats and in awake cats produced no increase in arterial pressure. In anesthetized cats, exchange transfusion with an albumin solution reduced hematocrit from 30 to 18%, increased cerebral blood flow, and dilated pial arterioles. In contrast, reducing hematocrit by transfusing the diafiltered polymer did not increase cerebral blood flow as pial arterioles constricted. These results are consistent with the hypothesis that the increase in arterial pressure associated with cell-free hemoglobin transfusion depends on hemoglobin extravasation. Constriction observed in the cerebrovascular bed with a nonextravasating hemoglobin polymer at low hematocrit is presumably a regulatory response to prevent overoxygenation at low blood viscosity.