Liposome-encapsulated hemoglobin (LEH) with a low oxygen affinity (l-LEH, P(50) = 45 mm Hg) was found to be protective in the rodent and primate models of ischemic stroke. This study investigated the role of LEH with a high O(2) affinity (h-LEH, P(50) = 10 mm Hg) in its protective effect on brain ischemia. The extent of cerebral infarction was determined 24 h after photochemically induced thrombosis of the middle cerebral artery from the integrated area of infarction detected by triphenyltetrazolium chloride staining in rats receiving various doses of h-LEH as well as l-LEH. Both h-LEH and l-LEH significantly reduced the extent of cortical infarction. h-LEH remained protective at a lower concentration (minimal effective dose [MED]: 0.08 mL/kg) than l-LEH (MED: 2 mL/kg) in the cortex. h-LEH reduced the infarction extent in basal ganglia as well (MED: 0.4 mL/kg), whereas l-LEH provided no significant protection. h-LEH provided better protection than l-LEH. The protective effect of both high- and low-affinity LEH may suggest the importance of its small particle size (230 nm) as compared to red blood cells. The superiority of h-LEH over l-LEH supports an optimal O(2) delivery to the ischemic penumbra as the mechanism of action in protecting against brain ischemia and reperfusion.

An artificial oxygen carrier, liposome-encapsulated hemoglobin (LEH), protective in a rodent stroke model, was quantitatively evaluated in monkeys. Serial positron emission tomography studies using the steady-state 15 O-gas inhalation method were performed to quantify O 2 metabolism, which was compared based on the infarction extent and immunohistochemical evaluation in 19 monkeys undergoing middle cerebral artery occlusion (3 h), infusion of various LEH doses (n ϭ 11), empty liposome (n ϭ 4), or saline (n ϭ 4) 5 min after the onset of ischemia, and reperfusion for 5 h. There was no significant difference in O 2 metabolism until 3 h after reperfusion, when the cerebral metabolic rate of O 2 (CMRO 2 ) was significantly less suppressed in the cortex [mild suppression in CMRO 2 (71-100%) of preischemic ipsilateral control as in the ischemic penumbra: 64.7 Ϯ 14.3% in empty liposome versus 32.4 Ϯ 7.9% in LEH (2 ml/kg) treatment, P Ͻ 0.05] but not in basal ganglia. Immunohistochemical studies showed a reciprocal expression of microtubular-associated protein II expression in the cortex and LEH deposition in basal ganglia, suggesting the LEH perfusion, but not deposition, afforded the protection. Doseresponse studies revealed that as little as 0.4 ml/kg LEH (24 mg/kg hemoglobin) was effective in preserving CMRO 2 , whereas 2 and 10 ml/kg were protective in significantly reducing the area of infarction as well, by 66 and 56%, respectively, compared with animals receiving saline. CMRO 2 and histological integrity were better preserved early after 3-h occlusion and reperfusion of the middle cerebral artery of monkeys receiving LEH early after onset of ischemia.Although ischemic stroke is a major cause of morbidity and mortality in developed societies (Broderick et al., 1998), medical attention is often delayed because of low public awareness of stroke symptoms (Williams et al., 1997). Increased risk of hemorrhagic transformation (Lapchak, 2002) limits the therapeutic time window and makes it more difficult to apply reperfusion therapy (NINDS rt-PA Stroke Study Group, 1995), as with coronary circulation. Although cell-free hemoglobin has been proposed to reduce ischemic injury, the associated toxicities have limited its utility (Natanson et al., 2008). Liposome-encapsulated human hemoglobin (LEH) (Ogata Y, 2000) has been developed as an artificial O 2 carrier that is functionally and structurally similar to red blood cells (RBCs). The lipid bilayer of encapsulated hemoglobin prevents renal clearance, extravasation, and direct contact of hemoglobin with vascular smooth muscle (Ogata, 2001), thus prohibiting vascular constriction, prolonging the in vivo halflife of LEH (Kaneda et al., 2009), and reducing the dosage required for beneficial effects (Kawaguchi et al., 2007). Although liposomes are much smaller (230 nm), their O 2 -carrying capacity is equivalent to human RBCs under room air respiration (Kaneda et al., 2009). These physical characteristics prompted us to examine the therapeutic potential of LEH, not as an...

To evaluate the energy metabolism of peripheral skeletal muscle during exercise in patients with chronic respiratory impairment, the 31P-nuclear magnetic resonance (NMR) spectra of forearm muscle were investigated in nine patients and nine age-matched control subjects. We calculated the phosphocreatine (PCr) to PCr + inorganic phosphate (PI) ratio, the time constant of PCr recovery and the intracellular pH. The exercise consisted of repetitive hand grips against a 2-kg load every 3 s for 6 min (0.33 W). The patients showed a marked decrease in the PCr/(PCr + PI) ratio and pH in the muscle during exercise in contrast to the control subjects whose PCr/(PCr + PI) showed a minor decrease without any change in pH. The relationship between PCr utilization and pH demonstrated that anaerobic glycolysis switched on earlier in patients with chronic respiratory impairment. A split PI peak was observed in five of nine patients during exercise. The PCr/(PCr + PI) ratio during the last minute of exercise correlated significantly with the vital capacity (% predicted), with the FEV1/FVC, with the body weight, with the maximum strength of hand grip, and with the muscle mass. The results indicate impaired oxidative phosphorylation and the early activation of anaerobic glycolysis in the muscles of patients with chronic respiratory impairment. Several factors related to chronic respiratory impairment, such as disuse, malnutrition and dysoxia, would contribute to the metabolic changes observed in the muscles examined.

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Background and Purpose-Liposome-encapsulated hemoglobin (LEH; TRM-645) is a novel oxygen (O 2 ) carrier with a lower O 2 affinity (P 50 O 2 ϭ40 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. Methods-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. Results-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. Conclusions-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.