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Intraosseous Transfusion With Liposome-Encapsulated Hemoglobin Improves Mouse Survival After Hypohemoglobinemic Shock Without Scavenging Nitric Oxide

Shono, Satoshi*†; Kinoshita, Manabu; Takase, Bonpei; Nogami, Yashiro§; Kaneda, Shinichi; Ishihara, Masayuki; Saitoh, Daizoh**; Kikuchi, Makoto††; Seki, Shuhji

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doi: 10.1097/SHK.0b013e3181e46e93

Abstract

INTRODUCTION

Hemoglobin-based oxygen carriers (HBOCs) might be very useful for resuscitation of patients with fatal hypohemoglobinemic shock, especially in a prehospital setting where conventional blood transfusion is not available. Hemoglobin-based oxygen carriers have many advantages, such as long shelf life, not needing refrigeration and cross-matching, and reducing the risk of iatrogenic infection (1-3). Nevertheless, several clinical trials have reported that resuscitation from hemorrhagic shock using HBOCs does not seem to be very effective (4-7). We believe that one of the reasons for these unfavorable results is that hemoglobin (Hb) molecules of HBOCs might easily bind to NO when released into the vasculature, because the HBOCs used in the clinical trials had no lipid membrane, being termed cell-free Hb (8). Natanson et al. (7) have pointed out that this NO scavenging effect might result in systemic vasoconstriction, decreased blood flow, increased proinflammatory mediators and potent vasoconstrictors, and a loss of platelet inactivation, creating conditions that may lead to vascular thrombosis of the heart or other organs. Therefore, development of a novel cellular HBOC that prevents direct contact between Hb and NO is an attractive goal that would limit the effects of NO scavenging.

Liposome-encapsulated Hb (LEH) was developed at the Terumo Research and Development Center (Terumo Co, Tokyo, Japan) (9, 10). Unlike cell-free Hb, LEH has a unique structure, with Hb encapsulated within a lipid bilayer membrane (liposome)-mimicking human red blood cells (RBCs)-and thereby suppressing direct contact between the Hb and NO (11). Although LEH has a lipid membrane similar to that of natural RBCs, the vesicle size of LEH is 220 nm in diameter, which is smaller than a natural RBC (9). Recently, we demonstrated that LEH transfusion is capable of rescuing rats from lethal progressive hemodilution by improving tissue hypoxia (12). In that study, we also demonstrated that LEH transfusion does not decrease the plasma nitrogen oxide levels in rats, suggesting that LEH does not have a potent NO scavenging effect. However, we did not examine the plasma NO2 or NO3 levels precisely.

In the case of prehospital hemorrhagic shock patients, it is in practice difficult to put a catheter into the peripheral vessels, because these vessels are collapsed from massive blood loss and centralization of circulating blood. Alternative transfusion routes that do not collapse even in hemorrhagic shock are necessary for effective and prompt resuscitation. The intraosseous route may provide a useful means of rapidly establishing vascular access, because intraosseous infusion has been a rapid, reliable method of achieving vascular access under emergency condition in children (13-15). Therefore, we have developed the administration of a cellular-type HBOC, LEH, via the intraosseous route, and we here report that intraosseous infusion with the LEH can effectively rescue mice from hypohemoglobinemic shock without scavenging plasma NO2 or NO3.

MATERIALS AND METHODS

Animal preparation

This study was conducted according to the guidelines of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College, Japan. Male C57BL/6 mice (7 weeks old, 20-23 g; SLC Japan Inc, Hamamatsu, Japan) were studied.

Preparation of LEH, washed mouse RBCs, and 5% albumin solution

The LEH (TRM-645) was prepared at the Terumo Research and Development Center (Terumo Co, Tokyo, Japan). Briefly, purified human Hb solution was prepared from outdated human RBCs provided by the Japanese Red Cross, with inositol hexaphosphate as an allosteric effector, nicotinamide adenine dinucleotide as a coenzyme, and glucose, adenine, and inosine as substrates. After washing, human RBCs were hemolyzed with simultaneous virus inactivation. Subsequently, Hb was concentrated using a reverse osmosis membrane and sterilized. Thereafter, the purified Hb was adjusted to 40 to 50 mmHg of P50 (the oxygen partial pressure at which Hb is half saturated with oxygen) by adding inositol hexaphosphate. After adjusting the P50, purified Hb was encapsulated with lipid ingredients through the use of high-speed emulsification. The surface of the encapsulating lipid membrane was then modified with 5-kd polyethylene glycol. The LEH was diluted with saline to achieve a final Hb concentration of 6 g/dL and then deoxygenated with N2 bubbling for storage. The diameter of the LEH was approximately 220 nm. The total lipid concentration of the LEH solution was 3.9 g/dL, with the methemoglobin proportion at 6.3%. The content of lipopolysaccharide was less than 0.1 EU/mL (9, 10).

To prepare washed mouse RBCs, blood was taken from nontreated mice under ether anesthesia and added to citrate phosphate dextrose solution, which contained 2.63 g sodium citrate, 2.32 g glucose, 327 mg citric acid, and 251 mg sodium biphosphate per 100 mL.

The preservation time was less than 3 days. Before use, the preserved blood was centrifuged at 3,000g for 10 min at 4°C, washed three times with 0.9% saline, and diluted with saline to achieve a final concentration of 6 g/dL because RBC concentration has been determined to set equal oxygen transporting efficiencies for LEH and mouse RBCs (12).

Before use, human serum albumin (HSA; Kaketsuken, Kumamoto, Japan) was added to both the LEH and mouse RBC suspensions to achieve a final albumin concentration of 5 g/dL (colloid osmotic pressures, 20 mmHg) (16). Addition of HSA allows us to exclude osmotic effects during correction of hypovolemia, because precise evaluation is needed of the effects of the reagents on hypohemoglobinemic shock. The colloid infusions are usually more effective than crystalloids in restoring myocardial blood flow and oxygen transport after acute hemorrhage (17). A 5% HSA solution (5 g/dL) was also prepared.

Hypohemoglobinemic shock by blood exchange and intravenous fluid resuscitation

Under ether anesthesia, a plastic catheter was put into the mouse superior vena cava. Thereafter, 0.2 mL of blood was withdrawn, and immediately, isovolumetric 5% albumin was administered via catheter. This blood exchange was repeated until the mice died to establish a lethal hypohemoglobinemic shock model. No mice could survive beyond 8 such blood exchanges. Therefore, after 7 exchanges of blood, the mice received intravenous administration with 0.5 mL of LEH, 5% albumin solution, or washed RBCs.

The effect of additional intravenous LEH or RBC transfusion after first intravenous transfusion

The mice received intravenous administration with 0.5 mL of LEH (n = 20) or washed RBCs (n = 10) 6 h after blood exchange and the first intravenous transfusion.

Intraosseous or intravenous fluid resuscitation following massive hemorrhage

To establish an intraosseous infusion route in mice, the femur was punctured with a 25-gauge needle followed by laparotomy under deep ether anesthesia. Immediately after infusion with indocyanine green (ICG; Daiichi, Tokyo, Japan) via this intraosseous route, the inferior vena cava was stained with ICG (indicated by arrow, Fig. 1), suggesting a rapid flow into the systemic circulation.

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Fig. 1:
Indocyanine green was administered by intraosseous infusion via the right femur. The circle shows the abdominal inferior vena cava, which was stained with ICG (arrow).

To produce hemorrhagic shock in mice, a 27-gauge needle was put into the femoral vein, and 0.8 mL of blood was withdrawn. The mean arterial pressure (MAP) showed approximately 40 mmHg. Subsequently, the femur (opposite side) was punctured with a 25-gauge needle, and then, 1 mL of 5% albumin was administered intraosseously. This intraosseous administration with albumin effectively rescued the subject mice. Five minutes after initial resuscitation, 0.3 mL of blood was additionally withdrawn, and subsequently, 1 mL of LEH, 5% albumin, or washed RBCs was also administered via the intraosseous route or the intravenous route (femoral vein).

Measurement of Hb in mouse blood samples containing LEH and RBC

Hemoglobin concentration in LEH could not be accurately determined, because the liposome capsules interfered with the spectrophotometric measurement of Hb absorbance. However, the measured Hb concentration in the LEH solution, as determined by the Erma PCE 170 hematology analyzer, was 5-fold higher than the actual concentration that was determined when using a specific enzyme-linked immunosorbent assay for human Hb during the production of LEH (see Figure, Supplemental Digital Content 1, https://links.lww.com/SHK/A51). We then estimated the actual Hb concentration in the LEH-transfused mouse samples based on the measured Hb concentration of each sample. First, the mouse RBC-derived Hb concentration in an LEH-mixed blood sample was estimated by reproducing an RBC suspension that had the same hematocrit as the sample. We then measured its Hb concentration, because the hematocrit in the LEH-mixed blood sample reflected the concentration of the mouse RBCs, given that the mean corpuscular volume essentially remained unchanged in all of the mice during the experiment. The LEH-derived Hb concentration was then obtained by subtracting the mouse RBC-derived Hb concentration from the measured Hb concentration, with the determination of the actual LEH-derived Hb concentration calculated by dividing the obtained LEH-derived Hb concentration by 5. The total concentration of Hb in the samples containing a mixture of RBCs and LEH was determined by adding the estimated LEH-derived Hb concentration and the mouse RBC-derived Hb concentration (12).

Measurements of plasma NO2, NO3, TNF, and erythropoietin levels

Collected blood samples were immediately heparinized and then centrifuged at 50,000g at 4°C for 20 min to remove the LEH particles. The plasma supernatant was stored at −80°C until assay. For NO determination, the plasma was mixed with methanol (1:1) followed by centrifugation at 10,000g at 4°C for 10 min to remove proteins, and the supernatant was used for measurement of plasma NO2 and NO3 levels. Determinations of plasma NO levels were performed on a high-performance liquid chromatography-Griess system (ENO-20; Eicom, Kyoto, Japan). This instrument applied a post-column derivatization method with Griess reagent for nitrite and nitrate detection. At first, nitrite and nitrate are separated from the other substances on a separation column. Thereafter, nitrite reacts with the Griess reagent and generates diazo compounds. The separated nitrate is reduced by a cadmium-copper column to react with the Griess reagent. The level of diazo compounds is then measured by a visible detector installed in a column oven at high stability and sensitivity. The detection limits and sensitivities were 0.01 μM for both NO2 and NO3. The loading volume of plasma was 10 μL. The area under the curve of each chromatogram was calculated for quantitative analysis of NO2 and NO3, using the analyzing software PowerChrom (AD Instrument Co, Ltd, Tokyo, Japan).Plasma samples were also tested with enzyme-linked immunosorbent assay kits for TNF (BD Pharmingen) and mouse Epo immunoassay (R&D Systems).

Operation procedure and samples analysis

All operations are performed with a microscope (SZ6045; Olympus Optical, Tokyo, Japan) and microsurgical technique. If the failure of the first fluid resuscitation or technical problems were found, the samples were removed from the data. The reason of failure was mainly unexpected bleeding or extravasation such as intramuscular infusion or subcutaneous infusion.

Statistical analysis

Statistical analyses were performed using the Stat View 4.02J software package (Abacus Concepts, Berkeley, Calif). Survival rates were compared by the Wilcoxon signed rank test. Statistical evaluations between two groups were compared using the Student t test, and any other statistical evaluations were compared using one-way ANOVA, followed by the Bonferroni post hoc test. Data are presented as the mean ± SE. P < 0.05 was considered to be statistically significant.

RESULTS

Fatal hypohemoglobinemia induced by blood exchange

Blood exchange was performed on mice (n = 15) by a 0.2-mL blood withdrawal and an isovolumetric intravenous injection of 5% albumin. The mice repeatedly received this blood exchange until they died. No mice survived beyond eight iterations of this blood exchange (Fig. 2A). Mean arterial pressure was decreased to 40 mmHg in the mice after seven exchanges of blood (Fig. 2B), and Hb concentration was decreased to approximately 5 g/dL because of progressive hemodilution, suggesting a fatal hypohemoglobinemia (Fig. 2C). Hematocrit, RBC count, platelet, and WBC counts were also decreased with hemodilution (Fig. 2, D-G).

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Fig. 2:
Data on the survival (A), mean arterial pressure (B), Hb level (C), hematocrit (D), RBC count (E), WBC count (F), and platelet count (G) of mice undergoing progressive hemodilution. The mice repeatedly had 0.2 mL of blood withdrawn followed by isovolumetric intravenous injection with 5% albumin. Data are mean ± SE from 15 mice.

Resuscitation of fatal hypohemoglobinemia by intravenous transfusion with LEH or RBCs

After seven exchanges of blood, the mice were intravenously transfused with 0.5 mL LEH (n = 20), 5% albumin (n = 20), or washed RBCs (n = 20). Although no mice were rescued by albumin transfusion, all of the LEH- and RBC-transfused mice recovered from hypohemoglobinemia within 6 h (normally fatal), suggesting a success in initial resuscitation (Fig. 3A). Both transfusions with LEH and RBCs promptly restored MAP to 70 mmHg from 30 mmHg. Nevertheless, the survival rate of LEH-transfused mice gradually decreased to 25% at 36 h, whereas RBC-transfused mice sustained a higher survival rate (Fig. 3A). Both LEH-transfused and RBC-transfused mice showed an increase in Hb concentration immediately after transfusion (at 5 min) (Fig. 3B). However, LEH-transfused mice did not show an increase in hematocrit or RBC count after transfusion (although RBC-transfused mice did), because the present hematology analyzer cannot detect a vesicle the size of LEH (200 nm) (Fig. 3, C and D). White blood cell count was transiently increased in the mice immediately after RBC transfusion but not after LEH transfusion (Fig. 3E). The platelet count was also increased 24 h after RBC transfusion but not after LEH transfusion (Fig. 3F). These findings suggest the possibility that hematologic alterations induced by massive blood transfusion do not occur after massive LEH transfusion.

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Fig. 3:
The effect of resuscitation fluid on the survival (A), Hb level (B), hematocrit (C), RBC count (D), WBC count (E), and platelet count (F) in mice with fatal hypohemoglobinemia. Fatal hypohemoglobinemia was induced in the mice by seven 0.2-mL exchanges of blood. Thereafter, the mice were treated by intravenous transfusion with 0.5 mL LEH, 5% albumin, or washed RBCs. Data are mean ± SE from 20 mice in each group. *P < 0.05 vs. the othergroups.

Serum TNF and plasma NO levels after resuscitation by intravenous transfusion with LEH or RBCs

Serum TNF levels were increased in the mice with repeated blood exchanges, whereas those levels decreased after transfusion with either RBCs or LEH (Fig. 4A); no statistical difference in the change in TNF levels was observed between the two groups. Although plasma NO2 levels were not increased in the mice during the blood exchanges, they were promptly increased after RBC transfusion. Plasma NO2 levels also gradually increased in the mice after LEH transfusion, but the increase was quite different from that after RBC transfusion (Fig. 4B). However, no obvious decrease in the plasma NO2 levels was observed after LEH transfusion. Plasma NO3 levels were slightly decreased in both groups not only during the blood exchanges but also after RBC transfusion (Fig. 4C). These findings suggest that LEH transfusion does not induce a potent NO scavenging effect in the subject mice.

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Fig. 4:
The change in serum TNF (A), plasma NO2 (B), and NO3 (C) levels in the mice during the blood exchanges and after resuscitation by LEH or washed RBC transfusions, as described in Figure 2. Data are mean ± SE from 10 mice in each group. *P < 0.05 vs. the other group.

The effect of additional intravenous LEH or washed RBC transfusion after first intravenous transfusion

We examined the effect of additional intravenous LEH transfusions (n = 20) or washed RBC transfusions (n = 10) 6 h after first transfusion. Twenty percent of the mice died within 6 h and did not receive LEH or washed RBC transfusion. The remained mice all survived after an additional washed RBC administration. The survival of mice receiving an additional LEH transfusion extended to 24 h, but the survival rate had decreased to same level as a single LEH administration at 48 h (Fig. 5).

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Fig. 5:
Survival of repeated LEH transfusion and RBC transfusion after LEH transfusion in mice with fatal hypohemoglobinemia. Fatal hypohemoglobinemia was induced in the mice by seven 0.2-mL exchanges of blood. Thereafter, the mice were treated by intravenous transfusion with 0.5 mL LEH or washed RBCs 6 h after intravenous transfusion with 0.5 mL LEH. Data are mean ± SE in each group. *P < 0.05 vs. the other groups.

Resuscitation of massive hemorrhage model by intraosseous transfusion with LEH or RBCs

Mice had 0.8 mL of blood withdrawn and subsequently were intraosseously infused with 1 mL of 5% albumin. All subjected mice survived the first hemorrhage with this treatment, although no mice survived without any fluid resuscitation. Next, those mice had 0.3 mL of additional blood withdrawn and then were intraosseously transfused with 1 mL of either LEH (n = 10), 5% albumin (n = 10), or RBCs (n = 10). Immediately after the second withdrawal, Hb concentration was decreased to approximately 6 g/dL in the subject mice, suggesting a severe hypohemoglobinemia. Intraosseous infusion with 5% albumin rescued approximately 30% of the subjected mice after the second withdrawal; however, intraosseous transfusion with LEH significantly increased mouse survival (Fig. 6A). Unexpectedly, intraosseous transfusion with RBCs did not effectively increase mouse survival (Fig. 6A). These results suggest the following two important findings. First, intraosseous transfusion with RBCs may not be effective for serious massive hemorrhage in mice. Second, intraosseous transfusion with LEH might be very effective for even such a serious hemorrhage.

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Fig. 6:
The effect of intraosseous transfusion on the survival (A), Hb level (B), hematocrit (C), and RBC count (D) in mice. The mice had 0.8 mL of blood withdrawn followed by intraosseous infusion with 1 mL of 5% albumin. Subsequently, they had 0.3 mL of additional blood withdrawn. Thereafter, the mice were treated by intraosseous transfusion with 1 mL of either LEH, 5% albumin, or washed RBCs. Data are mean ± SE from 10 mice in each group. *P < 0.05 vs. the other groups.

Consistent with survival rates, intraosseous transfusion with LEH significantly increased Hb concentration in the mice compared with either 5% albumin or RBCs (Fig. 6B), suggesting that LEH can effectively flow into the systemic circulation from the femur, unlike RBCs. Intraosseous transfusion with either LEH or 5% albumin increased neither RBC count nor hematocrit in the mice 1 h after transfusion, although intraosseous transfusion with RBCs gave such an increase (but slight) (Fig. 6, C and D), because the hematology analyzer cannot detect the LEH vesicles.

Serum TNF, erythropoietin, and plasma NO levels after intraosseous transfusion

Intraosseous transfusion with RBCs increased serum TNF levels in mice immediately after the transfusion (at 5 min) and kept them higher levels, in contrast to intraosseous transfusion with LEH or 5% albumin (Fig. 7A). Because intraosseous transfusion with LEH tended to increase hematocrit and RBC count beyond 48 h after the transfusion (Fig. 6, C and D), we examined the effect of LEH on the induction of erythropoietin, which stimulates RBC production in the bone marrow. Erythropoietin levels were increased after intraosseous transfusion and peaked at 12 h in all three groups. However, there was no significant difference in the erythropoietin levels among the three groups (Fig. 7B), suggesting that LEH does not affect erythropoietin-stimulated RBC production in the bone marrow. No significant decreases in the plasma NO2 or NO3 levels were observed in the mice after intraosseous transfusion with LEH relative to levels with 5% albumin or RBCs (Fig. 7C and D), suggesting that LEH does not have a potent NO scavenging effect, similar to our results for intravenous transfusion.

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Fig. 7:
The change in serum TNF (A), erythropoietin (B), plasma NO2 (C), and NO3 (D) levels in mice after intraosseous transfusion. The mice had blood withdrawn and intraosseous infusion as described in Figure 5. The changes in serum/plasma mediators after intraosseous transfusion with LEH, 5% albumin, or washed RBCs are shown. Data are mean ± SE from 10 mice in each group. *P < 0.05 vs. the other groups.

Resuscitation of massive hemorrhage model by intravenous transfusion with LEH or RBCs

Finally, we examined the effect of intravenous transfusion with LEH or RBCs on the mouse survival in this hemorrhage model. After the second blood withdrawal, the mice were intravenously transfused with LEH (n = 10), 5% albumin (n = 10), or RBCs (n = 10). As expected, intravenous transfusion with RBCs effectively increased mouse survival as well as that with LEH (Fig. 8A). Consistently, intravenous transfusion with RBCs significantly increased Hb concentration, hematocrit, and RBC count in the mice after transfusion (Fig. 8, B-D), suggesting an effective and rapid replenishment of RBC in the systemic circulation.

F8-8
Fig. 8:
The effect of intravenous transfusion on the survival (A), Hb level (B), hematocrit (C), and RBC count (D) in mice. The mice had 0.8 mL of blood withdrawn from the femoral vein and intraosseous infusion with 1 mL of 5% albumin. Subsequently, they had 0.3 mL of additional blood withdrawn. Thereafter, the mice were treated by intravenous transfusion with 1 mL of either LEH, 5% albumin, or washed RBCs. Data are mean ± SE from 10 mice in each group. *P<0.05 vs. the other groups.

DISCUSSION

Intraosseous transfusion with LEH more effectively rescued mice from fatal hemorrhage without scavenging NO than did intraosseous transfusion with RBCs. The present study clearly demonstrates the advantage of LEH, relative to conventional blood transfusion, as a blood substitute in resuscitation from massive hemorrhage in prehospital environment.

Despite many physiological modifications of Hb (2), cell-free Hbs reportedly increased the risk of myocardial infarction and death in a meta-analysis of the data from the clinical trials (7). These disadvantages of cell-free Hbs are thought to derive from their vasopressor effect, resulting from NO scavenging (2, 9, 10). Thus, NO scavenging induced by cell-free Hbs must be one of the most serious and fundamental adverse effects of the resuscitation of patients with hemorrhagic shock. Cross-linkage and/or polymerization of cell-free Hbs seem to be insufficient to prevent NO scavenging, because basically they do not have a structure like a cellular membrane, which may be indispensable in preventing direct contact between Hb and NO.

It is noteworthy that the current encapsulated Hb induced neither an NO scavenging effect nor elevation of serum TNF in mice after either intravenous or intraosseous transfusion. Mice receiving intravenous or intraosseous transfusion with LEH showed no marked decrease in the plasma NO2 or NO3 levels. Although the mice transfused with RBCs also showed no decrease in NO3 levels, NO2 levels were promptly increased after intravenous RBC transfusion. Regarding this, it has been reported that blood pressure decreased after isovolemic exchange transfusion with RBCs, in which increased blood viscosity induced shear stress-mediated production of NO in endothelial cells, leading to vasodilatation and hypotension (18-20). We speculate that a similar mechanism may explain the increased NO production in the first RBC infusion model. This is a first report measuring the plasma NO2 and NO3 levels after HBOC transfusion and, more importantly, showing a significant protection from both NO2 and NO3 scavenging effects induced by HBOC. Our novel HBOC, namely LEH, might have a potential for rescuing hemorrhagic shock patients without NO scavenging.

The intraosseous infusion route is reported to be a viable means of blood transfusion in a nonhemorrhagic swine model (21). Crystalloid infusion via the intraosseous route with a fixed (not rapid) rate was as effective as peripheral and central intravenous accesses for reversible hemorrhagic shock (22). However, the tortuous vascular architecture in the bone marrow might cause substantial hydraulic resistance to rapid infusion with fluid. The flow volume of intraosseous infusion with RBCs was significantly smaller than for saline under both normal gravitational and 300-mmHg pressures (23). Thus, rapid transfusion with RBCs via the intraosseous route may not be effective in rescuing the host from hemorrhagic shock. Therefore, crystalloid or colloid fluid infusion via intraosseous route is usually used for hypovolemic shock. However, asanguineous fluid cannot rescue the patients from fatal severe anemia resulting from massive Hb loss and tissue hypoxia (12), and administration of an oxygen carrier including LEH or RBCs is preferable for resuscitation in that situation.

Thus, we tried to establish the simple model with intraosseous infusion after blood exchange. However, this model did not succeed because approximately 90% of the animals were dropped because of anesthetic problems, unexpected cardiac arrest, or bleeding when attempting the intraosseous route. The second model tried to reproduce resuscitation in a clinical situation when another hemorrhage occurs after adequate asanguineous infusion for an initial hemorrhage. Whereas approximately 32% of the original Hb remained after blood exchange and transfusion in the first dilution model, 40% of original Hb remained in the second model, which may explain the fact that the mouse survival after intraosseous albumin infusion was better than intravenous albumin transfusion.

Intraosseous transfusion with LEH significantly increased the survival of and the Hb levels in mice above those seen with RBCs. Because the size of the LEH vesicle is about 220 nm, which is 1/30 to 1/40 the size of a natural mouse RBC, the influx of LEH from the femur to the systemic circulation might be better than RBC influx, thereby improving mouse survival. Furthermore, the biconcave shape, size, and deformability of natural RBCs might be appropriate for passing quickly through capillaries, but perhaps not through the tortuous vascular architecture in the bone marrow. The round shape, smaller size, and elastic hardness of LEH vesicles may be more suitable for the intraosseous route. Extravasation of infusions and compartment syndrome occasionally occur in intraosseous infusion and may be associated with major morbidity. When we intraosseously transfused RBC into the mice at high pressure, extravasation of RBCs was sometimes observed around the inserting site by pressure infusion. Extravasation occurred more frequently after RBC transfusion than after LEH transfusion. Interestingly, TNF levels after intraosseous RBC transfusion were higher than those after LEH or 5% albumin treatment. Because washed RBCs cannot pass through the tortuous vascular architecture in the bone marrow smoothly, activated bone marrow macrophages may phagocytose trapped RBCs and produce TNF.

As expected, intravenous transfusion with RBCs effectively rescued the mice from fatal hypohemoglobinemia, whereas intravenous transfusion with LEH eventually could not rescue more than half of the mice (although it could rescue most of them within several hours). Some investigators have pointed out similar problems, in that the effective period of cell-free Hb is not very long (24). A clinical report on a bovine-derived Hb solution (HBOC-201; Hemopure, OPK Biotech, Cambridge, Mass) in South Africa has demonstrated that some patients presented severe anemia again 24 to 36 h after the initial administration to relieve life-threatening symptoms of anemia (24). Although mice intravenously transfused with LEH showed a change in Hb levels similar to that of mice transfused with RBCs, their survival rates were quite different. Liposome-encapsulated hemoglobin may lose its function as an oxygen carrier soon after transfusion. The functional half-life of LEH has been reported as approximately 6 h in mice (25). Although the present LEH has a long circulation time achieved by modification of the liposomal surface with hydrophilic polymer, it was reported that the functional half-life (the period to a reduction by half of actual oxygen transport efficiency) seems to be shorter than natural Hb half-life because of methemoglobin generation (10, 25, 26). Methemoglobin formation might be one of the reasons why the LEH-transfused mice showed lower survival rates than did RBC-transfused mice despite similar Hb levels. However, transfusion with LEH saved mice for 6 h, although no mice were rescued by albumin transfusion. If survival is extended beyond 6 h, the possibility of the medical team rescuing the patient increases. Red blood cell transfusion after resuscitation with LEH may improve the survival of hypohemoglobinemic shock patients. In addition, the half-life of LEH in rat and monkey was about 10 and 70 h, respectively; therefore, a longer half-life of LEH could be anticipated in humans (10).

The present study indicates a need for transfusion with an oxygen carrier after conventional fluid resuscitation in situations of uncontrolled massive hemorrhage that can lead to fatal hypohemoglobinemia. In a hospital environment, intraosseous infusion is not needed except in special situations, because the medical staff can insert a central venous catheter for transfusion. In a prehospital environment such as a battlefield or in a confined space in disaster situations, however, sometimes only the intraosseous route will be available. If LEH can be administrated to patients via the intraosseous route, resuscitation using oxygen-carrying fluid can be performed under critical conditions without special training or medical expertise.

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Keywords:

Blood substitute; hemodilution; hemorrhage; resuscitation; NO2; NO3; TNF; erythropoietin

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