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.

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.

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 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.

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.

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.

Liposome-encapsulated hemoglobin accelerates skin wound healing in mice.

Effects of liposome-encapsulated hemoglobin with high O2 affinity (m-LEH, P50O2 = 17 mm Hg) on skin wound healing in mice were examined. Two full-thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in Balb/c mice. Two days later (day 2), the animals randomly received intravenous m-LEH (2 mL/kg, n = 12), homologous blood transfusion (red blood cell [RBC], n = 11), or saline (n = 12). The same treatment was repeated 4 days after wounding (day 4), and the sizes of the skin defects and ulcers were monitored on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. While the size of the skin defect in relation to the stent ring remained the same in all groups, the size of the ulcer compared with the skin defect (or silicone stent) became significantly reduced on days 4 and 7 in mice treated with m-LEH (46 ± 10% of pretreatment size, P < 0.01) compared with mice treated with RBC transfusion (73 ± 6%) or saline (76 ± 7%). m-LEH treatment significantly accelerated granulation, increased epithelial thickness, suppressed early granulocyte infiltration, and increased Ki67 expression in accordance with the ulcer size reduction, while there was no difference in surface blood flow or CD31 expression among the groups. The results suggest that m-LEH (2 mL/kg) may accelerate skin wound healing in Balb/c mice via mechanism(s) involving reduced inflammation and increased metabolism, but not by improved hemodynamics or endothelial regeneration.

Liposome-encapsulated hemoglobin improves energy metabolism in skeletal muscle ischemia and reperfusion in the rat.

The effect of liposome-encapsulated hemoglobin (LEH) was tested in a rodent model of limb ischemia and reperfusion--causing local reperfusion injury and a cascade of systemic responses. Intracellular pH (pHi) and phosphocreatine (PCr)/inorganic phosphate (Pi) ratio were serially monitored using ³¹P-nuclear magnetic resonance spectroscopy with a 2-cm solenoid coil on a rodent hind limb. After baseline measurements, the right hind limb underwent ischemia for 70 min, followed 10 min later by intravenous administration of LEH (10 mL/kg, n = 6), homologous red blood cells (RBCs, n = 6), saline (n = 6), or no treatment (n = 6). Reperfusion was then observed for an additional 60 min. While pHi decreased precipitously after the onset of ischemia and even following reperfusion, LEH-treated rats had significantly milder intracellular acidosis compared with all other groups during ischemia, and after reperfusion as well throughout the observation with the saline-treated rats. In contrast, the PCr/Pi ratio decreased regardless of treatment after ischemia until reperfusion, when the ratio returned toward normal or the energy status improved only in the LEH-treated rats, while the ratio remained depressed in the control animals receiving RBC, saline, or no treatment. Morphological studies 7 days later revealed a tendency toward suppressed mononuclear cell infiltration with preservation of muscular mass and structure in the LEH-treated rats. LEH treatment after early limb ischemia appeared to improve intracellular energy metabolism and eventually preserve skeletal muscle in a rodent model of limb ischemia and reperfusion.

Effect of liposome-encapsulated hemoglobin on antigen-presenting cells in mice.

Liposome-encapsulated hemoglobin (LEH) is removed from the circulation and degraded in the reticuloendothelial system, including dendritic cells (DCs) and macrophages. Therefore, LEH at a large dose may overload the system, cause a competitive inhibition in antigen-presenting activity, and impair the immune response of the host. Changes in cellularity of immunocompetent cells were monitored serially up to 4 weeks by flow cytometry in wild-type mice receiving 20 mL/kg of LEH, syngeneic red blood cells (RBCs), or saline. DCs were collected from the host spleen 1, 7, and 28 days after receiving the solution and were cocultured with naïve cluster of differentiation 4 T cells from T-cell receptor transgenic mice in the absence or presence of third-party antigens. After LEH administration, the cellularity of DCs and macrophages in the recipient spleen remained unchanged from control mice receiving RBCs or saline. While subset populations and costimulatory molecule expressions were different, DCs from LEH-administered mice expressed high levels of interleukin-2 production and helper T-cell activation in response to a third-party antigen and superantigens, as did the DCs from control mice receiving RBCs or saline. The results suggest that 20 mL/kg of LEH does not greatly alter antigen-presenting activity to third-party antigens.

Liposome-encapsulated hemoglobin ameliorates tumor hypoxia and enhances radiation therapy to suppress tumor growth in mice.

We hypothesize that liposome-encapsulated hemoglobin with high O2 affinity (P5002 = 12 mm Hg, h-LEH) may increase O2 delivery to hypoxic tumors and enhance radiation therapy synergistically to suppress tumor growth. First, h-LEH (5, 10, and 20 mL/kg) was intravenously infused 30 min before radiation (20 Gy) of SCCVII tumor grown in C3H/HeN mice. Second, 10 mL/kg of h-LEH was administered 30, 60, 90, and 120 min prior to radiation to determine optimal timing. Tumor size was monitored thereafter to titrate tumor growth suppression. Third, additional mice with SCCVII tumor were infused with h-LEH or empty liposome (EL), and tumors were excised at various time points for immunohistochemical examination of h-LEH and hypoxia-inducible factor-1α (HIF-1α). h-LEH was most effective at 10 mL/kg in comparison to 5 or 20 mL/kg of h-LEH or EL. Tumor growth was most suppressed when the interval between h-LEH infusion and radiation was shortest, 30 min. As a result, 10 mL/kg of h-LEH infusion 30 min prior to radiation prolonged 5-fold tumor-growth time from 20.0 days (radiation and EL) to 26.5 days, P<0.01, synergy ratio 1.42. While human hemoglobin (h-LEH) was detected in tumors 0.5 to 24 h after administration, HIF-1α accumulation was sparse and became significantly reduced compared to controls 48 and 72 h after h-LEH infusion. h-LEH (10 mL/kg) was highly effective in enhancing radiation therapy synergistically under ambient respiration against tumor growth in mice. Decreased accumulation of HIF-1α in h-LEH-treated tumor may suggest targeted tumor oxygenation as a potential mechanism.

Liposome-encapsulated hemoglobin alleviates hearing loss after transient cochlear ischemia and reperfusion in the gerbil.

To test liposome-encapsulated hemoglobin (LEH) in transient cochlear ischemia/reperfusion as a model of sudden deafness, Mongolian gerbils were randomly assigned to receive 2 mL/kg of either low-affinity LEH (l-LEH, P5002 = 40 mm Hg), high-affinity LEH (h-LEH, P5002 = 10 mm Hg), homologous red blood cells (RBCs), or saline (each group n = 6) 30 min before 15-min occlusion of the bilateral vertebral arteries and reperfusion. Sequential changes in hearing were assessed by auditory brain response 1, 4, and 7 days after ischemia/reperfusion, when the animals were sacrificed for pathological studies. h-LEH was significantly more protective than l-LEH in suppressing hearing loss, in contrast to RBC or saline treatment, at 8, 16, and 32 kHz, where hearing loss was most severe (P < 0.05 between any two groups) on the first day after cochlear ischemia/reperfusion. Thereafter, hearing loss improved gradually in all groups, with a significant difference among groups up to 7 days, when morphological studies revealed that the inner hair cells but not the outer hair cells, were significantly lost in the groups in the same order. The results suggest that pretreatment with h-LEH is significantly more protective than l-LEH in mitigating hearing loss and underlying pathological damage, in contrast to transfusion or saline infusion 7 days after transient cochlear ischemia/reperfusion.

Liposome-encapsulated hemoglobin ameliorates ischemic stroke in nonhuman primates: an acute study.

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 = 11), empty liposome (n = 4), or saline (n = 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 +/- 14.3% in empty liposome versus 32.4 +/- 7.9% in LEH (2 ml/kg) treatment, P < 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. Dose-response 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.

A Novel Composite Biomaterial Made of Jellyfish and Porcine Collagens Accelerates Dermal Wound Healing by Enhancing Reepithelization and Granulation Tissue Formation in Mice.

Background and Objective: Impaired dermal wound healing represents a major medical issue in today's aging populations. Granulation tissue formation in the dermis and reepithelization of the epidermis are both important and necessary for proper wound healing. Although a number of artificial dermal grafts have been used to treat full-thickness dermal loss in humans, they do not induce reepithelization of the wound, requiring subsequent epithelial transplantation. In the present study, we sought a novel biomaterial that accelerates the wound healing process. Approach: We prepared a composite biomaterial made of jellyfish and porcine collagens and developed a hybrid-type dermal graft that composed of the upper layer film and the lower layer sponge made of this composite biomaterial. Its effect on dermal wound healing was examined using a full-thickness excisional wound model. Structural properties of the dermal graft and histological features of the regenerating skin tissue were characterized by electron microscopic observation and immunohistological examination, respectively. Results: The composite biomaterial film stimulated migration of keratinocytes, leading to prompt reepithelization. The regenerating epithelium consisted of two distinct cell populations: keratin 5-positive basal keratinocytes and more differentiated cells expressing tight junction proteins such as claudin-1 and occludin. At the same time, the sponge made of the composite biomaterial possessed a significantly enlarged intrinsic space and enhanced infiltration of inflammatory cells and fibroblasts, accelerating granulation tissue formation. Innovation: This newly developed composite biomaterial may serve as a dermal graft that accelerates wound healing in various pathological conditions. Conclusion: We have developed a novel dermal graft composed of jellyfish and porcine collagens that remarkably accelerates the wound healing process.

Liposome-encapsulated hemoglobin reduces the size of cerebral infarction in rats: effect of oxygen affinity.

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.

Liposome-encapsulated hemoglobin alleviates brain edema after permanent occlusion of the middle cerebral artery in rats.

Liposome-encapsulated hemoglobin (LEH) was proven to be protective in cerebral ischemia/reperfusion injury. The present study evaluated LEH in a rat model of permanent middle cerebral artery (MCA) occlusion to clarify its effect during ischemia and reperfusion. Five minutes after thread occlusion of the MCA, rats were infused with 10 mL/kg of LEH (LEH, n = 13), and compared with normal controls (n = 11). Additional animals received the same MCA occlusion with no treatment (CT, n = 11), saline (saline, n = 10), empty liposome solution (EL, n = 13), or washed red blood cells (RBC, n = 7). Severity of brain edema was determined 24 h later by signal strength in T2-weighted magnetic resonance imaging of the cortex, striatum, hippocampus, and pyriform lobe. The results showed that brain edema/infarction observed in any vehicle-infused control was significantly more severe than in LEH-treated rats. There was a tendency toward aggravated edema in rats receiving ELs. LEH infusion at a dose of 10 mL/kg significantly reduced edema formation as compared to other treatments in a wide area of the brain 24 h after permanent occlusion of the MCA. Low oncotic pressure of EL and LEH solution (vehicle solution) appeared to cause nonsignificant aggravation of edema and reduced protective effects of LEH.

Effects of liposome-encapsulated hemoglobin on human immune system: evaluation in immunodeficient mice reconstituted with human cord blood stem cells.

As preclinical evaluation in animals does not necessarily portray human responses, liposome-encapsulated hemoglobin (LEH), an artificial oxygen carrier, was tested in immunodeficient mice reconstituted with human hematopoietic stem cells (cord blood-transfused NOD/SCID/IL-2R(gammanull)[CB-NOG] mice). Changes in immunocompetent T-cell and B-cell composition in peripheral blood, spleen, and bone marrow were examined 2 and 7 days after 10 mL/kg of intravenous administration of LEH, empty liposome (EL), or saline using immunohistochemical and flow cytometrical techniques in wild-type mice and CB-NOG mice. Responses to intraperitoneal administration of toxic shock syndrome toxin-1 (TSST-1) under the absence or presence of LEH (10 mL/kg) were also determined 4 h and 3 days later in terms of lymphocyte composition and IL-2 plasma level in wild-type as well as CB-NOG mice. When liposome (LEH or EL) was administered to wild-type or CB-NOG mice, the composition of B-cells and T-cells in the spleen or peripheral blood failed to show any consistent or significant changes. The responses to a bacterial antigen (TSST-1) measured by IL-2 production were comparable regardless of the presence or absence of LEH in wild-type as well as in CB-NOG mice. Cellularity, distribution, and maturation of these human cells in peripheral blood, spleen, and bone marrow were comparable among the groups. The results suggest that simple LEH administration may not change immune cellularity, and LEH presence may not largely affect the early T-cell response to bacterial enterotoxins in murine as well as in reconstituted human immune systems.