A synthetic porphyrin as an effective dual antidote against carbon monoxide and cyanide poisoning.

Simultaneous poisoning by carbon monoxide (CO) and hydrogen cyanide is the major cause of mortality in fire gas accidents. Here, we report on the invention of an injectable antidote against CO and cyanide (CN-) mixed poisoning. The solution contains four compounds: iron(III)porphyrin (FeIIITPPS, F), two methyl-ß-cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and imidazole (Im3CD, I), and a reducing agent (Na2S2O4, S). When these compounds are dissolved in saline, the solution contains two synthetic heme models including a complex of F with P (hemoCD-P) and another one of F with I (hemoCD-I), both in their iron(II) state. hemoCD-P is stable in its iron(II) state and captures CO more strongly than native hemoproteins, while hemoCD-I is readily autoxidized to its iron(III) state to scavenge CN- once injected into blood circulation. The mixed solution (hemoCD-Twins) exhibited remarkable protective effects against acute CO and CN- mixed poisoning in mice (~85% survival vs. 0% controls). In a model using rats, exposure to CO and CN- resulted in a significant decrease in heart rate and blood pressure, which were restored by hemoCD-Twins in association with decreased CO and CN- levels in blood. Pharmacokinetic data revealed a fast urinary excretion of hemoCD-Twins with an elimination half-life of 47 min. Finally, to simulate a fire accident and translate our findings to a real-life scenario, we confirmed that combustion gas from acrylic cloth caused severe toxicity to mice and that injection of hemoCD-Twins significantly improved the survival rate, leading to a rapid recovery from the physical incapacitation.

Artificial oxygen carrier improves fatigue resistance in slow muscle but not in fast muscle in a rat in situ model.

The effects of liposome-encapsulated hemoglobin with high O2 affinity (h-LEH), an artificial O2 carrier in skeletal muscle, were studied by in situ fatigue resistance test in fast-type plantaris (PLT) and slow-type soleus (SOL) muscles with or without ischemia in the rat. The distal tendons of PLT and SOL muscles were isolated in situ and individually attached to the force transducers to record the developed tension in response to stimuli (80 Hz tetanus train, 1.5 minutes) to the ipsilateral sciatic nerve. The fatigue resistance test (five sets separated by 2-minute rests) was evaluated in terms of tension attenuation (fatigue) from the initial to the last tension (A) during each set, attenuation of the initial (B) or last tension (C) in each set, as compared to the first set in the presence or absence of ischemia or h-LEH (10 mL/kg). While ischemia significantly enhanced fatigue only in PLT, h-LEH showed no effect regardless of the perfusion pattern (normal/ischemia) or muscle-type (PLT/SOL) during each set (A). In parameter (B), set-by-set fatigue development was observed in PLT, whereas h-LEH-SOL showed a trend of advanced fatigue resistance. Such trends became clear in the parameter C (last tension), because h-LEH-SOL exerted, rather than decreased, the tension enhancement regardless of the presence or absence of ischemia, whereas there were no h-LEH effects in PLT. In addition, faster recovery of the nicotinamide adenine dinucleotide content in the muscle after 10 minutes of all fatigue tests was observed in h-LEH-SOL, while saline-SOL still showed a significantly higher value than that of control. These results suggested that additional O2 supply by h-LEH may accelerate the tricarboxylic acid cycle/electron transport chain in slow-type aerobic SOL muscle containing abundant mitochondria and contribute to the faster removal of muscle fatigue substances such as lactate.

PEGylated carboxyhemoglobin bovine (SANGUINATE) ameliorates myocardial infarction in a rat model.

Artificial oxygen (O2 ) carriers were reported to be protective in ischemia/reperfusion (I/R) in various organs including the heart. In the current study, 20 rats underwent ligation (MI) of the left anterior descending artery, were treated with 10 mL/kg of PEGylated carboxyhemoglobin bovine (SANGUINATE, S+, n = 10) or saline (S-, n = 10) 10 minutes after MI and daily thereafter for 3 days, and were followed by weekly echocardiography for 4 weeks, when they had left ventricular pressure volume relationship (PVR) analyses followed by necropsy. Echocardiography showed an increase in end-systolic dimension rather than end-diastolic dimension, preserved fractional shortening (36 vs. 26%, P < .01), and milder mitral regurgitation in S+ compared with S- rats. PVR revealed a milder increase in end-systolic volume, larger stroke volume (101 vs. 74 µL, P < .005) and cardiac output (33.4 vs. 23.8 mL/min, P = .004) in S+ rats in actual determination and under a wide range of standardized loading conditions 4 weeks after MI. Excised heart showed significantly limited area of MI (8.9 vs. 13.3%, P = .028). The results suggest that SANGUINATE in short-term repeated doses may accelerate weight recovery, preserving the myocardium, mitral competence, and cardiac function after MI. The mechanism of action and optimal treatment for MI remain to be studied.

Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Liposome-Encapsulated Hemoglobin Improves Tumor Oxygenation as Detected by Near-Infrared Spectroscopy in Colon Carcinoma in Mice.

Liposome-encapsulated hemoglobin (LEH) with high (h-LEH, P50 O2  = 10 mm Hg) or low O2 affinity (l-LEH, P50 O2  = 40 mm Hg) may improve O2 delivery to sensitize tumor tissues for radiotherapy. A total of 10 mL/kg of h-LEH, l-LEH, red blood cells (RBCs), or saline was infused in mice transplanted with murine colon carcinoma with near-infrared spectroscopy (NIRS) detectors set at the tumor (right leg) and intact muscle (left leg). NIRS recorded changes in the amount of oxyhemoglobin (oxyHb), deoxyhemoglobin (deoxyHb), and their sum (tHb) with the animals spontaneously breathing room air (10 min), pure O2 (5 min), and then back to room air. The tumor was finally excised for histological examination. In mice treated with h-LEH, tHb significantly increased compared to mice receiving other solutions. The magnitude was significantly attenuated in the tumor compared to the intact muscle under room air. Reciprocal changes in oxyHb and deoxyHb between intact muscle and tumor in response to infused solutions allowed assumption of average tissue PO2 between 30 and 40 mm Hg in muscle and at around 10 mm Hg in tumor. While O2 respiration increased oxyHb and decreased deoxyHb both in muscle and tumor, their sum or tHb consistently decreased in muscle and increased in tumor regardless of preceding infusion. Such responses were totally reversed when mice were placed under hypoxia (10% O2 ), suggesting that a lack of physiological circulatory regulation in tumor may account for heavier immunohistochemical staining for human hemoglobin in tumors of mice treated with h-LEH than with l-LEH. The results suggest that h-LEH may cause significant tumor oxygenation compared to RBC, l-LEH, or saline probably due to its nanometer size (vs. RBC) and high O2 affinity (vs. l-LEH) without increasing O2 content in the intact tissue (vs. O2 respiration) probably due to a lack of physiological circulatory regulation.

Liposome-Encapsulated Hemoglobin Accelerates Skin Wound Healing in Diabetic dB/dB Mice.

Since liposome-encapsulated hemoglobin with high O2 affinity (h-LEH, P50 O2  = 10 mm Hg) has been reported to accelerate skin wound healing in normal mice, it was tested in dB/dB mice with retarded wound healing, as seen in human diabetics. Two full-thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in dB/dB mice. Two days later (day 2), the animals were randomly assigned to receive intravenous h-LEH (2 mL/kg, n = 7) or saline (2 mL/kg, n = 7). The same treatment was repeated 4 days after wounding (day 4), and the size of the skin lesions was analyzed by photography, surface perfusion was detected by Laser-Doppler imager, and plasma cytokines and chemokines were determined on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. The size of the ulcer compared to the skin defect or silicone stent became significantly reduced on days 4 and 7 in mice treated with h-LEH (47 ± 8% of original size), similar to the level in wild-type mice, compared to saline-treated dB/dB mice (68 ± 18%, P < 0.01). Mice treated with h-LEH had significantly attenuated inflammatory cytokines, increased surface perfusion, and increased Ki67 expression on day 7 in accordance with the ulcer size reduction, while there was no significant difference in chemokines, histological granulation, epithelial thickness, and granulocyte infiltration detected by immunohistochemical staining in the ulcer between the treatment groups. The results suggest that h-LEH (2 mL/kg) early after wounding may accelerate skin wound healing in dB/dB mice to levels equivalent to wild-type mice probably via mechanism(s) involving reduced hypoxia, increased surface perfusion, suppressed inflammation, accelerated in situ cell proliferation and protein synthesis.

Effect of Oxygen Affinity of Liposome-Encapsulated Hemoglobin on Cerebral Ischemia and Reperfusion as Detected by Positron Emission Tomography in Nonhuman Primates.

We tested a hypothesis that liposome-encapsulated hemoglobin (LEH) with high oxygen (O2 ) affinity (h-LEH, P50 O2 = 10 mm Hg) may work better than LEH with low O2 affinity (l-LEH, P50 O2 = 40 mm Hg) in cerebral ischemia and reperfusion injury using positron emission tomography (PET) in primates undergoing middle cerebral artery (MCA) occlusion and reperfusion. Cerebral blood flow (CBF), O2 extract fraction (OEF), and cerebral metabolic rate of O2 (CMRO2 ) were successively determined by PET before ischemia, at 2 h of ischemia, immediately after reperfusion, and 3 h after reperfusion. Five minutes after MCA occlusion, 10 mL/kg of h-LEH (n = 6) was intravenously infused and compared with the results from previous data of monkeys treated with l-LEH (n = 6), empty liposome (n = 4), or saline (n = 8) as control. After the series of PET studies, the integrated area of cerebral infarction was determined histologically in 12 coronal brain slices. There was no significant difference in CBF, OEF, or CMRO2 up to 2 h of ischemia. A high CBF with a low OEF tended to be suppressed after reperfusion in LEH-treated monkeys. Three hours after reperfusion, the area of mild CMRO2 reduction (down to -30%) decreased (P < 0.05) and the area of mild CMRO2 increase (up to 30%) expanded in LEH-treated monkeys (P < 0.05) regardless of O2 affinity with no difference in the area of moderate-to-severe reduction (<-30%) or increase (<+30%) in CMRO2 compared to animals treated with empty liposome or saline. Distribution of CMRO2 reduction and histological damages showed that LEH mainly protected the cerebral cortex rather than basal ganglia where neuronal dendritic processes were severely lost. There was little difference between the animals treated with l-LEH or h-LEH both at 10 mL/kg or between treatment with empty liposome or saline. In conclusion, LEH was effective regardless of O2 affinity in preserving CMRO2 and in reducing the area of histological damage in the cerebral cortex, but not in basal ganglia, shortly after occlusion/reperfusion of MCA in monkey.

Preoperative Risk Levels and Vascular Access in Transcatheter Aortic Valve Implantation-A Single-Institute Analysis.

Although transcatheter aortic valve implantation (TAVI) has been indicated for patients with high surgical risk, indications for or against the procedure become more difficult as vascular access becomes more proximal and/or invasive in order to accommodate patients with even higher risks. We compared preoperative factors including the European System for Cardiac Operative Risk Evaluation (EuroSCORE) and Society of Thoracic Surgeons Predicted Risk of Mortality (STS) score with postoperative survival in 195 patients undergoing TAVI during 2.5 years (January 2010 to June 2012), when vascular access routes were developed from iliofemoral (IL/Fm access, n = 149), axillo-clavicular, apical, and direct aortic approaches (alternative access, n = 46). Logistic regression analyses showed that alternative access was associated with reduced 30-day survival (P = 0.024), while high surgical risk (>15% in both EuroSCORE and STS score) was associated with reduced 1-year survival (P = 0.046). Thus, patients treated via IL/Fm access had acceptable outcome regardless of preoperative risk levels while patients with low surgical risk (<15%) had favorable outcome irrespective of access route. Since the remaining patients with combined risk factors, high preoperative risk level (>15%) requiring alternative access, had a prohibitive risk in our experience, they might have been considered untreatable or not amenable even to TAVI and offered medical or alternative managements.

Ventricular conduction defects after transcatheter aortic valve implantation: a single-institute analysis.

Patients with aortic stenosis tend to develop ventricular conduction problems, which are known adverse events following transcatheter aortic valve implantation (TAVI). Changes in ventricular conduction status after TAVI were analyzed in 195 consecutive patients from a single institute registered in FRANCE2 between February 2010 and June 2012. Among the 195 patients, 29 had a prior pacemaker implantation (+PM) and 6 had acute catastrophic hemodynamics that made a full electrocardiogram (ECG) unavailable. Among the remaining 160, PM was newly required in 28 (17.5%, PM+) but not in 132 (PM-), which included 21 (13.1%) who developed new left bundle branch block (BBB), 12 (7.5%) had right BBB, and 99 (61.9%) had no change. While PM requirement had no correlation with preoperative factors, there was significant association with the development of right BBB with Edwards Sapien/XT (P = 0.003), and new left BBB (P = 0.012) and complete heart block requiring PM with CoreValve (22.6% vs. Edwards Sapien/XT, 7.4%, P = 0.016). Whereas postoperative survival regarding PM status (+PM, PM+, PM-), vascular access, valve size or type showed no difference, delayed heart block (n = 12, Day 2 or later) was associated with poor survival (P = 0.038) compared with the remaining PM+ patients with earlier onset (n = 16, Day 0 or 1). As a result, PM+ patients (n = 28) had significantly longer intensive care unit (ICU) stay and hospitalization than PM- or +PM patients. The results suggest that ventricular conduction problems requiring PM occurred more frequently after TAVI (17.5%) than with usual surgical replacement. Nonetheless, conduction problems failed to influence postoperative survival for up to 3 years on average with use of PM, but therefore did increase medical costs.

Usefulness of myoglobin containing cobalt heme cofactor in designing a myoglobin-based artificial oxygen carrier.

The structure and reactivity of cobalt-replaced myoglobin (Mb) were investigated to explore its possible application as an artificial oxygen carrier. Ligand binding analysis with relaxation kinetics revealed that various ligands bind to Co(III) Mb, contrary to the earlier thoughts. The equilibration process, however, was so slow that it proceeded over 90 min. These characteristic profiles of oxidized Co(III) Mb were ascribed to the electronic structure of Co(III) ion which is one electron larger than Fe(III) ion. The oxygen affinity of reduced Co(II) Mb was much smaller than that of Fe(II) Mb indicating that Co(II) Mb has excellent oxygen transport ability. The latter observation, together with the lack of carbon monoxide binding in Co(II) Mb, suggests utility of Co(II) Mb as Mb-based oxygen carriers. The present results on cobalt-substituted Mb are useful in designing myoglobin-based oxygen carriers.

Liposome-encapsulated hemoglobin accelerates bronchial healing after pneumonectomy in the rat with or without preoperative radiotherapy.

Liposome-encapsulated hemoglobin (LEH) has been reported to accelerate wound healing in the stomach and skin in an experimental setting. LEH was tested in bronchial anastomotic healing after radiation and pneumonectomy in the rat. Sprague-Dawley rats (n = 61) received preoperative radiation (20 Gy) to the chest and underwent left pneumonectomy with bronchial stump closure using the Sweet method 4 days later, when they were randomized to receive intravenous infusion of LEH with high O2 affinity (P50 O2 = 17 mm Hg, 10 mL/kg, n = 32) or saline (n = 29). Additional rats (n = 18) were treated in the same way without preoperative radiation. Bronchial anastomotic healing was evaluated 2 days after surgery by determining the bursting pressure and infiltration of neutrophils, monocytes, and macrophages. Bronchial bursting pressure was elevated in the rats receiving LEH both in the unirradiated group (LEH 212 ± 78 vs. saline 135 ± 63 mm Hg, P < 0.05) and in rats with preoperative radiation (LEH 162 ± 48 vs. saline 116 ± 56 mm Hg, P < 0.01). Moreover, the percentage of rats with bursting pressure <100 mm Hg tended to be smaller in the unirradiated group (LEH 1/9 [11.1%] vs. saline 4/9 [44.4%], NS) and was significantly reduced in irradiated animals (LEH 3/32 [9.4%] vs. saline 11/29 [38%], P < 0.05). There were no morphological differences except for macrophage infiltration to the anastomotic area, which was significantly prominent in the LEH-treated rats (P < 0.05) regardless of the presence or absence of preoperative irradiation (IR). The results suggest that LEH with high O2 affinity may improve mechanical strength and morphological findings in bronchial anastomosis in rats regardless of the presence or absence of preoperative IR. The irradiated rats later treated with LEH had equivalent or better bronchial healing than that of saline-treated naïve animals undergoing pneumonectomy alone.

Effects of liposome-encapsulated hemoglobin on learning ability in tokai high-avoider rat after total brain ischemia and reperfusion.

Liposome-encapsulated hemoglobin with low O2 -affinity (l-LEH) was shown to be protective in focal brain ischemia and reperfusion (I/R) in rats and primates. We tested l-LEH in the transient whole brain ischemia in the Tokai high-avoider rat (THA), which has been selected, mated, and bred over 77 generations for a high and consistent learning ability determined by the Sidman avoidance test (SAT). Young/naïve (before SAT) and adult/parent (after SAT) THA rats underwent acute and complete four-vessel occlusion in the chest for 3 or 5 min, administration of 2 mL/kg of l-LEH, saline, or homologous washed red blood cells (RBCs), reperfusion, and resuscitation. One week later, all rats underwent SAT, open-field behavioral observation, Morris water maze tests, and morphological study. Whereas young/naïve rats treated with l-LEH retained a rapid and consistent learning curve as in nonischemic controls, THA rats treated with RBCs or saline had retarded learning response on SAT as well as reduced cellularity in the amygdala. Adult/parent rats with established memory on SAT maintained perfect achievement even after I/R. In contrast, l-LEH-treated rats showed no better performance on Morris water maze (function) or cellularity of the CA1 sector of the hippocampus (morphology) compared with the rats treated with RBCs. Although task performance on SAT and Morris water maze appeared antithetical, morphological observations corresponded to the respective functions, suggesting that l-LEH was protective only for the amygdala on SAT tasks but not for the CA1 sector of the hippocampus on spatial orientation as in our previous studies on focal brain I/R, where the cortex was preserved better than basal ganglia.

Liposome-encapsulated hemoglobin enhances chemotherapy to suppress metastasis in mice.

Liposome-encapsulated hemoglobin with high O2 -affinity (P50 O2 = 10 mm Hg, h-LEH) was reported to enhance tumor radiosensitivity. We hypothesize that targeted O2 delivery to tumor hypoxia by h-LEH may also enhance chemotherapy to suppress tumor growth and metastasis in mice. Doxorubicin (DXR; 0.5 or 2 mg/kg i.p.) or S-1 (4 or 8 mg/kg orally) alone or in combination with h-LEH (5 mL/kg i.v.) was administered for 2 weeks to C57BL/6N mice inoculated with Lewis Lung Carcinoma (LLC) in the leg. After the 2-week therapy in six treatment groups, mice were sacrificed for quantitative assessment of tumor growth and lung metastasis. The tumor was then evaluated for its expression of hypoxia-inducible factor-1α (HIF-1α) and matrix metallopoteinase-2 (MMP-2) activity. Combined use of h-LEH and chemotherapeutic agents (DXR or S-1) showed no additional enhancement on suppression of the tumor growth over the chemotherapeutic agent alone. However, the combination use of h-LEH significantly suppressed the number and total area of metastatic colonies in the lung compared with each chemotherapeutic agent alone. Although HIF-1α expression and MMP-2 activity in the original tumor was significantly suppressed in the groups of mice treated with either DXR or S-1 alone, the addition of h-LEH to either agent showed further enhancement of oxygen-mediated degradation of HIF-1α and suppression of MMP-2 activity. Although the addition of h-LEH to DXR or S-1 had little effect on original LLC tumor growth, it significantly enhanced suppression of lung metastasis in mice.

Effects of liposome-encapsulated hemoglobin on gastric wound healing in the rat.

Liposome-encapsulated hemoglobin (LEH) may improve microcirculation and oxygen (O2 ) metabolism at a surgical wound to accelerate its healing. Ten mL/kg of LEH with high (h-LEH) or low O2 -affinity (l-LEH), homologous red blood cells (RBC), empty liposome or saline as a control was infused before a 10-mm incision and interrupted suture closure of the gastric wall in a total of 110 rats. Two and 4 days later, the stomach was excised for bursting pressure determination and histological sampling. The dose-response relationship was examined in 70 additional rats receiving progressively reduced doses of h-LEH. Hypoxia-inducible factor-1α (HIF-1α) was stained immunohistochemically in 54 other rats to examine its accumulation at the anastomotic sites. Bursting pressure of the surgical wound was significantly higher 2 days after surgery only in the h-LEH-treated rats (P < 0.05), but not at 4 days after surgery, when other rats showed increased bursting pressure to a nonsignificant level. Histological examination revealed less granulocyte infiltration, better granulation, and more macrophage infiltration in h-LEH-treated rats at 2 days, but no longer at 4 days postsurgery. Dose-response study revealed that 0.4 mL/kg of h-LEH (hemoglobin 24 mg/kg) was effective for elevating bursting pressure at 2 days. h-LEH-treated rats had significantly suppressed HIF-1α accumulation in the wound 6, 24, and 48 h after surgery as compared with control animals treated with homologous RBC or saline. In conclusion, the results suggest that h-LEH, but not l-LEH or homologous transfusion, may accelerate wound healing early after gastric incision and anastomosis in the rat. The mechanism(s) appears to be related to improved O2 supply, aerobic metabolism, and suppressed inflammation in the wound.

Real-time trafficking of PEGylated liposomes in the rodent focal brain ischemia analyzed by positron emission tomography.

Aliposomal drug delivery system was previously applied to ischemic brain model rats for the treatment of brain ischemia, and we observed that 100-nm-sized liposomes could extravasate and accumulate in the ischemic brain region even when cerebral blood flow was markedly reduced in permanent middle cerebral artery occlusion (p-MCAO) model rats. In the present study, we investigated the real-time cerebral distribution of polyethylene glycol (PEG)-modified liposomes (PEGliposomes) labeled with 1-[18F]fluoro-3,6-dioxatetracosane in p-MCAO rats by positron emission tomography (PET). [18F]-Labeled PEG-liposomes were intravenously injected into p-MCAO rats 1 h after the onset of occlusion, and then a PET scan was performed for 2 h. The PET scan showed that the signal intensity of [18F] gradually increased in the ischemic region despite the drastic reduction in cerebral perfusion, suggesting that PEG-liposomes had accumulated in and around the ischemic region. Therefore,drug delivery to the ischemic region by use of liposomes would be possible under ischemic conditions, and a liposomal drug delivery system could be a promising strategy for protecting the ischemic brain from damage before recovery from ischemia.

Nanoparticles accumulate in ischemic core and penumbra region even when cerebral perfusion is reduced.

The use of a liposomal drug delivery system is a promising strategy for avoiding side effects and enhancing drug efficiency by changing the distribution of the intact drug. We have previously shown that liposomal agents quickly accumulated in an ischemia-reperfusion region and ameliorated cerebral ischemia-reperfusion injury when they were injected after reperfusion in transient middle cerebral artery occlusion (t-MCAO) rats. In the present study, we hypothesized that liposomes also act effectively as a drug carrier in the ischemic state, since the integrity of the blood brain barrier is disrupted at an early stage after an ischemic event. To test this hypothesis, the cerebral distribution of fluorescence-labeled liposomes was observed in permanent MCAO (p-MCAO) rats. The liposomes accumulated in the ischemic core and the penumbra region when injected at 1 or 2h after occlusion. The accumulation in the ischemic core region was clearly greater than that in the penumbra region, despite the cerebral blood perfusion of the core region being substantially reduced. This result suggests that drug delivery to an ischemic region using liposomes is possible even when cerebral blood circulation has not recovered. Because liposomal drug delivery systems have the potential to effectively employ a number of agents that have failed in clinical trials, they may offer an effective strategy for achieving neuroprotection in stroke patients.

Relaxation analysis of ligand binding to the myoglobin reconstituted with cobaltic heme.

Myoglobin reconstituted with oxidized Co(III) deuteroheme was found to exhibit relatively large affinities to CN(-), N3(-), SCN(-), pyridine, and imidazole contrary to the early proposal. The ligand-induced changes in electronic spectra were less obvious than those of Fe(III) myoglobin owing to the absence of accompanied spin-state transition on the ligand binding. The relaxation kinetic analysis revealed that the ligand association rates were small and that the dissociation rates were still much smaller. The relatively large ligand affinities in Co(III) myoglobin were found due to the compensation of small association rates with fairly smaller dissociation rates. This is in marked contrast with the ligation profile in Fe(III) myoglobin where large association rates and small dissociation rates are generally observed. The rationale for the characteristic ligand-binding behavior of Co(III) myoglobin was provided on the basis of the properties of Co(III) which has an additional negative charge and forms stronger metal-ligand bonds than Fe(III).

Static cardiomyoplasty with synthetic elastic net suppresses ventricular dilatation and dysfunction after myocardial infarction in the rat: a chronic study.

Although static cardiomyoplasty prevents the left ventricle (LV) from dilatation, it may interfere with diastolic relaxation, or cause restriction. We developed a synthetic net with dual elasticity and tested its effect late after myocardial infarction in the rat. LV pressure-volume relationships (PVR) were successively analyzed before, after intravenous volume load, and 10 minutes after occlusion of the left anterior descending artery. Rats were then randomized into groups receiving synthetic net wrapping around the heart (NET+, n = 8) and only partially behind LV (NET-, n = 9), and they underwent the same PVR studies 6 weeks later. End-diastolic and end-systolic PVR were defined, and LV size and function were compared under standardized loading conditions. Although there was no difference in Day 0, increase in LV end-diastolic and end-systolic volumes were significantly attenuated in NET+ rats 6 weeks later when there was a significant correlation between LV volumes by PVR estimation and actual measurements, with significant differences in both measures between the groups: NET+ < NET-. The presence or absence of net did not show restrictive hemodynamics under acute volume load. Static cardiomyoplasty using a synthetic elastic net significantly attenuated LV dilatation and dysfunction without restriction late after myocardial infarction in the rat.

Liposome-encapsulated hemoglobin ameliorates ischemic stroke in nonhuman primates: longitudinal observation.

Liposome-encapsulated hemoglobin (LEH) is protective early after brain ischemia in rats and nonhuman primates, but it remains unclear whether the protection persists and confers any benefits beyond the acute phase of brain ischemia and reperfusion. Ten monkeys underwent middle cerebral artery occlusion, received LEH (2 mL/kg, n = 5) or saline (2 mL/kg, n = 5) 5 min later, and reperfusion 3 h later. Positron emission tomography studies were repeated for the cerebral metabolic rate of O2 (CMRO2 ) as well as glucose (CMRglc) up to 8 days after reperfusion, when the animals were euthanized for morphological studies. There was no difference in O2 metabolism until 3 h after reperfusion, when CMRO2 was significantly better preserved in the cortex, but not in basal ganglia, on Day 0 in LEH-treated monkeys. The extent of cortical infarction (saline 68 ± 10% vs. LEH 38 ± 9%, P < 0.05) and CMRO2 (mild suppression: saline 34 ± 10% vs. LEH 14 ± 4%, P < 0.05) remained significantly better preserved 8 days later, when CMRglc showed a similar pattern of cortical protection (mild suppression: saline 49 ± 15% vs. LEH 37 ± 4%, P < 0.05) in LEH-treated monkeys, together with regained body weight. Somatic weight control, morphological integrity, CMRO2 , and CMRglc were better preserved immediately, as well as 8 days after occlusion and reperfusion of the middle cerebral artery in monkeys receiving LEH early after onset of ischemia.