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Novel Hemoglobin-Based Oxygen Carrier Bound With Albumin Shows Neuroprotection With Possible Antioxidant Effects

Originally publishedhttps://doi.org/10.1161/STROKEAHA.118.021467Stroke. 2018;49:1960–1968

Abstract

Background and Purpose—

A hemoglobin-albumin cluster, 1 core of hemoglobin covalently bound with 3 shell albumins, designated as HemoAct was developed as a hemoglobin-based oxygen carrier. We aim to investigate neuroprotection by HemoAct in transient cerebral ischemia and elucidate its underlying mechanisms.

Methods—

Male rats were subjected to 2-hour transient middle cerebral artery occlusion and were then administered HemoAct transarterially at the onset of reperfusion. Neurological and pathological findings were examined after 24 hours of reperfusion to identify neuroprotection by HemoAct. Intermittent measurements of cortical blood flow and oxygen content were performed, and a histopathologic analysis was conducted on rats during the early phase of reperfusion to assess the therapeutic mechanism of HemoAct. In addition, the antioxidant effects of HemoAct were examined in hypoxia/reoxygenation-treated rat brain microvascular endothelial cells.

Results—

Neurological deterioration, infarct and edema development, and the activation of MMP-9 (matrix metalloprotease-9) and lipid peroxidation after 24 hours of reperfusion were significantly ameliorated by the HemoAct treatment. Reductions in blood flow and tissue partial oxygen pressure in the cortical penumbra after 6 hours of reperfusion were significantly ameliorated by the HemoAct treatment. The histopathologic analysis of the cortical penumbra revealed that HemoAct in HemoAct-treated rats showed superior microvascular perfusion with the mitigation of microvascular narrowing changes than autologous erythrocytes in nontreated rats. Although HemoAct extravasated into the ischemic core with serum protein, it did not induce an increase in serum extravasation or reactive oxygen species production in the ischemic core. In vitro experiments with rat brain microvascular endothelial cells revealed that HemoAct significantly suppressed cellular reactive oxygen species production in hypoxia/reoxygenation-treated cells, similar to albumin.

Conclusions—

HemoAct exerted robust neuroprotection in transient cerebral ischemia. Superior microvascular perfusion with an oxygen delivery capability and possible antioxidant effects appear to be the underlying neuroprotective mechanisms.

Introduction

Because several randomized controlled trials on endovascular thrombectomy for acute ischemic stroke provided clear evidence of therapeutic efficacy,14 therapeutic approaches to ischemic stroke have markedly changed. Endovascular thrombectomy achieved higher rates of angiographically demonstrated revascularization and provided better functional outcomes.5 Although high recanalization rates are favorable for tissue salvage, delayed recanalization after severe ischemia contributes to tissue injury based on microvascular perfusion disorders. Severe neurological disorders and poor outcomes because of ischemia/reperfusion (I/R) injury have increased the need for neuroprotective therapeutic strategies against I/R injury.

Among various neuroprotective therapeutic strategies, artificial oxygen carriers, mainly hemoglobin-based oxygen carriers (HBOCs), have been used in anticipation of improvements in microvascular perfusion, more efficient oxygen delivery, and increases in collateral flow.68 We previously demonstrated that liposome-encapsulated Hb, a cellular-type HBOC, reduced I/R injury in a rat transient middle cerebral artery occlusion (tMCAO) model.9,10 In the present study, we used a novel cell-free HBOC, 1 core of Hb covalently bound with 3 human serum albumin molecules, designated as HemoAct.11 Because HemoAct is covered with albumin shells, its surface net charge becomes negative and induces electrostatic repulsion against the endothelial surface, resulting in suppressed leakage through the endothelium. This property prevents marked elevations in blood pressure and promotes a long period of blood retention.12 HemoAct may also exert beneficial effects in the treatment of microvascular perfusion disorders based on the albumin characteristics of volume expander and antioxidant.13 We herein aim to investigate the neuroprotection of HemoAct and its underlying mechanisms in the rat tMCAO model.

Methods

The data that support the findings of this study are available from the corresponding author on reasonable request. A detailed description of Materials and Methods can be found in the online-only Data Supplement.

Animals

All animal experiment protocols were approved by the Animal Studies Ethics Committee at the Hokkaido University Graduate School of Medicine. All procedures used in the present study were performed in accordance with the institutional guidelines for animal experimentation and the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan. A total of 125 rats were subjected to experiments which are described in the online-only Data Supplement.

HemoAct

HemoAct is an HBOC and its physical property is described in the online-only Data Supplement.

tMCAO Model

Transient focal cerebral ischemia was induced by right MCAO using a silicone rubber-coated nylon filament. Detailed procedures are described in the online-only Data Supplement.

In Vivo Experimental Protocol

Analysis of Effects of HemoAct on tMCAO Rats After 24 Hours of Reperfusion

The effects of HemoAct on the neurological and pathological findings were investigated after 24 hours of reperfusion in the 4 (control, vehicle, 50% HemoAct, and HemoAct) groups. Detailed procedures are described in the online-only Data Supplement.

Analysis of Effects of HemoAct on tMCAO Rats During the Early Phase of Reperfusion

The effects of HemoAct on the cortical blood flow, tissue oxygen content, and microvascular perfusion were investigated during the early phase of reperfusion in the control and HemoAct groups. Detailed procedures are described in the online-only Data Supplement.

Neurological Scores

A neurological assessment was performed using an 18-point scale score. Detailed procedures are described in the online-only Data Supplement.

Evaluation of Brain Injury and Edema Volume

Infarct and edema volumes were evaluated using 2,3,5-triphenyltetrazolium chloride staining. Detailed procedures are described in the online-only Data Supplement.

Western Blotting

Western blotting was performed using anti–MMP-9 (matrix metalloproteinase-9) antibody and anti–4-hydroxynonenal (4-HNE) antibody. Detailed procedures are described in the online-only Data Supplement.

Cerebral Blood Flow Measurements

Cerebral blood flow was measured by Laser Doppler flowmetry in the middle cerebral artery territories. Detailed procedures are described in the online-only Data Supplement.

Tissue Partial Oxygen Pressure Measurement

Brain tissue partial oxygen pressure (Pto2) was measured by an oxygen electrode method. Detailed procedures are described in the online-only Data Supplement.

Immunohistochemical Staining

Immunohistochemical staining was performed with paraffin sections of the brain fixed in 4% paraformaldehyde. Detailed procedures are described in the online-only Data Supplement.

Analysis of Microvascular Perfusion and Narrowing Changes

Microvascular perfusion and narrowing changes were examined by immunohistochemical analysis in the control and HemoAct groups. Detailed procedures are described in the online-only Data Supplement.

Analysis of the Distribution of HemoAct and Its Related Effects in the Ischemic Core

The distribution of HemoAct, IgG, and 8-hydroxy-2′-deoxyguanosine in the ischemic core was examined by immunohistochemical analysis in the control and HemoAct groups. Detailed procedures are described in the online-only Data Supplement.

In Vitro Cellular Hypoxia-Reoxygenation Injury Model

The antioxidant effects of HemoAct were examined in hypoxia/reoxygenation-treated rat brain microvascular endothelial cells (RBMECs). Detailed procedures are described in the online-only Data Supplement.

Measurement of Reactive Oxygen Species Production in RBMECs

The measurement of reactive oxygen species (ROS) production was performed using 3 different methods: dihydroethidium fluorescent staining, an 8-hydroxy-2′-deoxyguanosine ELISA, and 4-HNE Western blotting. Detailed procedures are described in the online-only Data Supplement.

Analysis of Effects of Albumin on tMCAO Rats After 24 Hours of Reperfusion

The effects of albumin itself on tMCAO rats were examined and compared with the result in the analysis of the effects of HemoAct on tMCAO rats. Detailed procedures are described in the online-only Data Supplement.

Data Collection and Statistical Analysis

All data were collected by investigators blinded to the experimental groups and were presented as means±SD. Two group comparisons were performed by the Mann-Whitney U test. Multiple comparisons were conducted by a 1-way ANOVA followed by Bonferroni test or the Kruskal-Wallis test and then by the Steel-Dwass test. Sample sizes were selected based on our previous experiments. Values of P<0.05 were considered to be significant.

Results

Physiological Parameters

No significant differences were observed in the values of basic physiological parameters including blood gas and blood pressure before tMCAO or in the degree of cerebral blood flow (CBF) reductions during tMCAO between the 4 groups. Physiological parameters after the treatments were also not significantly different between the 4 groups, indicating that HemoAct did not cause adverse effects (Table II in the online-only Data Supplement).

Neurological Status and Infarct and Edema Volumes After 24 Hours of Reperfusion

The effects of HemoAct on the neurological status and infarct and edema volumes were investigated in the 4 groups (Figure 1A). No significant differences were observed in neurological scores between the 4 groups at the early phase of tMCAO or early phase of reperfusion (Figure 1B). However, the neurological status was significantly superior in the HemoAct group than in the control group (P<0.01), vehicle group (P<0.01), and 50% HemoAct group (P<0.05) after 24 hours of reperfusion (Figure 1B). Brain infarct volumes were significantly smaller in the HemoAct group (20.2±3.1%) and 50% HemoAct group (27.1±4.7%) than in the control group (55.2±3.6%; P<0.01) and vehicle group (53.2±4.3%; P<0.01; Figure 1C and 1D). The mean infarction volume of the HemoAct group was 37% that of the control group. Edema volumes were also significantly smaller in the HemoAct group than in the control group (P<0.01) and vehicle group (P<0.01; Figure 1D).

Figure 1.

Figure 1. Effects of HemoAct on neurological findings and brain infarct and edema volumes. A, Experimental design diagram. B, Neurological function evaluated using an 18-point scale at the early phase of middle cerebral artery occlusion (MCAO), the early phase of reperfusion, and 24 hours after reperfusion. *P<0.05 and **P<0.01. C, Representative images of brain sections with 2,3,5-triphenyltetrazolium chloride staining. D, Quantitative evaluation of brain infarct and edema volumes. **P<0.01. 50% H indicates 50% HemoAct group; C, control group; H, HemoAct group; and V, Vehicle group.

Activation of MMP-9 and Lipid Peroxidation After 24 Hours of Reperfusion

The effects of HemoAct on the activation of MMP-9 and lipid peroxidation were investigated by Western blotting of MMP-9 and 4-HNE. The immunoblot intensity of MMP-9 was significantly lower in the HemoAct group than in the control group (P<0.05; Figure 2A). The immunoblot intensity of 4-HNE was significantly lower in the HemoAct group than in the vehicle group (P<0.05) and control group (P<0.05; Figure 2B).

Figure 2.

Figure 2. Effects of HemoAct on MMP-9 (matrix metalloproteinase-9) and 4-hydroxynonenal (4-HNE) production. A, Representative image of MMP-9 Western blotting and quantitative evaluation of immunoblots (n=5 in each group). B, Representative image of 4-HNE Western blotting and quantitative evaluation of immunoblots (n=5 in each group). *P<0.01. 50% H indicates 50% HemoAct group; C, control group; H, HemoAct group; and V, Vehicle group.

CBF and Tissue Pto2 in the Cortical Penumbra During the Early Phase of Reperfusion

CBF and tissue Pto2 were measured intermittently from just before tMCAO to 6 hours after reperfusion in the cortical penumbra of the control and HemoAct groups (Figure 3A). CBF and Pto2 were reduced after 6 hours of reperfusion in the control group, which indicated postischemic delayed hypoperfusion (Figure 3B and 3C). However, the reductions observed in CBF and Pto2 were suppressed in the HemoAct group, resulting in significant differences (P<0.01) in the values of CBF and Pto2 between the control and HemoAct groups after 6 hours of reperfusion (Figure 3B and 3C).

Figure 3.

Figure 3. Effects of HemoAct on cerebral blood flow (CBF) and tissue partial oxygen pressure (Pto2). A, Experimental design diagram. B, Time course changes in CBF in the control group and HemoAct group. C, Time course changes in Pto2 in the control group and HemoAct group. **P<0.01. MCAO indicates middle cerebral artery occlusion.

Microvascular Perfusion in the Cortical Penumbra During the Early Phase of Reperfusion

The status of microvascular perfusion in the cortical penumbra during the early phase of reperfusion was examined in the control and HemoAct groups (Figure 4A). Microvessels filled with rat autologous erythrocytes immunostained with the anti-rat Hb antibody decreased in number as time elapsed in the control group. However, microvessels filled with HemoAct immunostained with the anti-human albumin antibody did not decrease in number in the HemoAct group (Figure 4B). A quantitative analysis showed that the total number of Rat-Hb–positive vessels in the control group was significantly lower after 2 hours (P<0.01) and 6 hours (P<0.01) of reperfusion than at 0 hours of reperfusion. The number of positive vessels was significantly greater (P<0.01) in the HemoAct group than in the control group after 2 and 6 hours of reperfusion (Figure 4C).

Figure 4.

Figure 4. Effects of HemoAct on microvascular perfusion. A, Experimental design diagram. B, Representative images of immunohistochemistry with an anti-rat Hb antibody in the control group and an anti-human serum albumin (HSA) antibody in the HemoAct group. Arrowheads show rat-Hb–positive microvessels in the control group and HSA-positive (ie, HemoAct positive) microvessels in the HemoAct group. Scale bar=10 µm. C, Quantitative evaluation of the number of rat-Hb–positive microvessels in the control group and HSA-positive microvessels in the HemoAct group. **P<0.01, significantly different from the control at 0 hours. ##P<0.01, significant difference between 2 groups at each time point. C indicates control group; and H, HemoAct group; and MCA, middle cerebral artery.

Microvascular Narrowing Changes in the Cortical Penumbra During the Early Phase of Reperfusion

Microvascular morphological changes in the cortical penumbra during the early phase of reperfusion were examined in the control and HemoAct groups with von Willebrand factor immunohistochemistry (Figure 5A). Microvascular narrowing with an enlarged perivascular halo was observed in both groups during reperfusion (Figure 5B). A quantitative analysis showed that the cross-sectional width of microvessels was significantly smaller, whereas the cross-sectional width ratio of the perivascular halo to microvessels was significantly larger in both groups after 2 to 6 hours of reperfusion than in the control group at 0 hours of reperfusion (Figure 5C and 5D). In comparisons of the cross-sectional width of microvessels and the cross-sectional width ratio of the perivascular halo to microvessels between the control and HemoAct groups, the former was significantly greater (P<0.01) and the latter was significantly smaller (P<0.01) in the HemoAct group than in the control group after 6 hours of reperfusion (Figure 5C and 5D).

Figure 5.

Figure 5. Effects of HemoAct on microvascular narrowing changes. A, Experimental design diagram. B, Representative images of von Willebrand factor immunoreactivity in the control group and HemoAct group. Scale bar=10 µm. C, Quantitative analysis of temporal changes in the cross-sectional width of microvessels. D, Quantitative analysis of temporal changes in the cross-sectional width ratio of microvessels to the perivascular halo. **P<0.01, significantly different from the control at 0 hours. ##P<0.01, significant difference between 2 groups at each time point. C indicates control group; H, HemoAct group; and MCA, middle cerebral artery.

Distribution of HemoAct and Its Related Effects in the Ischemic Core During the Early Phase of Reperfusion

The distribution of HemoAct and its related effects on serum extravasation and ROS production in the ischemic core during the early phase of reperfusion was examined in an immunohistochemical analysis. HemoAct clearly extravasated into the ischemic core after 2 and 6 hours of reperfusion in most of the HemoAct-treated rats (Figure IIA in the online-only Data Supplement). HemoAct extravasation was sometimes accompanied by the extravasation of serum IgG (Figure IIA in the online-only Data Supplement). The intensity grade of IgG immunohistochemistry was somewhat, but not significantly, lower in HemoAct-treated rats than in control rats (Figure IIB in the online-only Data Supplement), suggesting that the extent of serum IgG extravasation in HemoAct-treated rats was similar or less than that in control rats. ROS production evaluated by 8-hydroxy-2′-deoxyguanosine immunohistochemistry was occasionally observed in control rats (2 out of 8 rats), but not in HemoAct-treated rats (0 out of 8 rats) (Figure IIA in the online-only Data Supplement). Therefore, HemoAct extravasation did not cause an increase in serum extravasation or ROS production in the ischemic core.

Effects of HemoAct on ROS Production in Cultured RBMECs Treated With Hypoxia-Reoxygenation

The antioxidant characteristics of HemoAct were examined in cultured RBMECs treated with hypoxia-reoxygenation. We used 2 hours of 1% O2 hypoxia and 6 hours of normoxic reoxygenation for the experimental conditions and conducted 4 treatments (control, Hb, albumin, and HemoAct) on RBMECs during the reoxygenation period (Figure 6A). Three assays to evaluate ROS production, dihydroethidium fluorescent staining, an 8-hydroxy-2′-deoxyguanosine ELISA, and 4-HNE Western blotting, showed similar results in this experiment. Although ROS production in the Hb group was similar to that in the control group, it was significantly lower in the albumin and HemoAct groups than in the control group, suggesting that HemoAct has similar antioxidant characteristics to albumin (Figure 6B through 6D).

Figure 6.

Figure 6. Effects of HemoAct on reactive oxygen species (ROS) production in cultured rat brain microvascular endothelial cells (RBMECs) treated with hypoxia-reoxygenation. A, Experimental design diagram. B, Representative fluorescent images of ROS production in RBMECs evaluated by dihydroethidium (DHE) staining (×400, Scale bar=50 μm) and quantitative evaluation of fluorescent intensity. C, Quantitative evaluation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) production. D, Representative image of 4-hydroxynonenal (4-HNE) Western blotting and quantitative evaluation of immunoblots. *P<0.05 and **P<0.01. Alb indicates albumin group; C, control group; H, HemoAct group; and Hb, hemoglobin group.

Discussion

HBOCs were originally studied and developed as a substitute for blood transfusion for blood loss because of injury and surgery. However, because HBOCs were found to have better and faster oxygenation capabilities than erythrocytes,14,15 the therapeutic potential of HBOCs in ischemic diseases has been actively examined.68,16 These studies demonstrated that HBOCs were beneficial for providing sufficient microcirculation and ameliorating I/R injury. We also previously reported that the infusion of liposome-encapsulated Hb exerted neuroprotective effects with superior microvascular perfusion in a rat tMCAO model.9,10 In line with these findings, we showed that HemoAct ameliorated neurological disorders and brain infarction because of I/R injury in the present study. Therefore, HBOCs have therapeutic potential for ischemic diseases with microvascular perfusion disorders.

Postischemic microvascular perfusion disorders after delayed recanalization are complex and sometimes exhibit contradictory responses, namely hyperperfusion or hypoperfusion.1719 Hyperperfusion is considered to increases in permeability and cellular extravasation, leading to the formation of edema and hemorrhagic transformation.20,21 On the other hand, hypoperfusion sometimes occurs after hyperperfusion, resulting in secondary ischemia because of a microvascular perfusion disturbance. This phenomenon has been known as the no-reflow phenomenon, which was demonstrated as microvascular occlusion by leukocyte adhesion, platelet accumulation, and fibrin formation in a primate transient ischemic model.22 However, another potential cause of microvascular perfusion disturbances was recently demonstrated in electron microscopic studies. Microvessels were transiently compressed and narrowed by swollen astrocyte end-feet after several hours of reperfusion, which may reduce microvascular perfusion.23,24 In the present study, we observed delayed postischemic hypoperfusion, represented as reductions in cortical CBF and Pto2 and a decrease in the appearance of erythrocytes in microvessels, which showed narrowing changes with an enlarged perivascular space after 6 hours of reperfusion. Considering the characteristics of HBOCs with small particle sizes, the conditions observed with microvascular narrowing changes in the I/R region are suitable for treatments with HBOCs to rescue brain tissue from irreversible damage.

To date, many HBOCs under development2528 have not yet been approved for clinical use because they cannot avoid increasing blood pressure because of the capture of nitric oxide by Hb. On the other hand, HemoAct covered with albumin shells has shown no increase of blood pressure in normal animals12 and even in the I/R injury model in the present study. On the basis of the characteristics of albumin, such as a negative net charge and high electrostatic repulsion, HemoAct may prevent to capture nitric oxide and increase blood pressure. In addition, albumin exerts neuroprotective effects on cerebral I/R injury because of its functions as a volume expander and antioxidant.29,30 As shown in Figure III in the online-only Data Supplement, 20% albumin, which was a comparable total protein mass concentration of albumin (20%=20 g/dL) to that in the administrated HemoAct (20 g/dL), alone moderately reduced infarction volumes in the same I/R injury model. Therefore, it is reasonable to consider HemoAct to have additive neuroprotective effects over other HBOCs. In the present study, we demonstrated the suppression of ROS production in in vivo and in vitro experiments (Figures 2B and 6). Therefore, HemoAct has an advantage for the treatment of ischemic diseases among HBOCs.

Albumin, which is either endogenous or exogenous, is extravasated into parenchyma and is taken up by neurons in rodent transient ischemic models.31,32 Although it has been unclear whether the extravasation and neuronal uptake of albumin are directly related to its neuroprotective effects, the administration of exogenous albumin exerted apparent neuroprotection without a change in the degree of IgG extravasation.31 This finding is similar to the present result showing that HemoAct extravasation did not cause an increase in IgG extravasation in the ischemic core, suggesting that HemoAct extravasation is a phenomenon based on albumin characteristics and does not enhance the deterioration of microvascular integrity and parenchymal cell viability. Although there are concerns that extracellular Hb results in an oxidative stress condition and neurotoxicity,33 HemoAct extravasation observed in the present study was not associated with ROS production, at least during the examination period (Figure II in the online-only Data Supplement; Figure 2B). Therefore, HemoAct extravasation in the ischemic core does not seem to have any adverse effects.

It is important to clarify how to apply HemoAct in stroke clinical practice. The experimental model we used in the present study is similar to patients who are treated with endovascular thrombectomy and receive transarterial drug infusions through a catheter after recanalization of the occluded artery. The reason why we selected a transarterial drug infusion was because we aimed to deliver HemoAct to postischemic tissue as rapidly and at as high a volume as possible. On the basis of the results of the present study, the transarterial HemoAct infusion had no adverse effects.

The limitations of the present study are as follows. The follow-up period was limited to the acute phase, up to 24 hours of reperfusion. One reason for the time limitation was the high death rate in this model after >24 hours of reperfusion. In future preclinical studies, long-term follow-ups will be needed using a less severe ischemic model. The next limitation is a lack of information on glucose levels, which is related to reperfusion state in stroke models (vascular effects of hyperglycemia) and could be 1 reason for the poor reperfusion. Another limitation is that experiments with different ischemic models, such as a permanent ischemic model, and different administration routes, including an intravenous infusion, need to be performed to expand indications for the HemoAct treatment. Furthermore, the long-term safety of HemoAct not only in normal animals but also in animals with cerebral ischemia is another issue that needs to be clarified.

In conclusion, HemoAct exerted strong neuroprotective effects on short-term I/R injury without Hb adverse effects. Superior microvascular perfusion and O2 transport as well as possible antioxidant effects seem to be the underlying neuroprotective mechanisms against I/R injury. HemoAct has potential as an HBOC in the treatment of ischemic diseases.

Acknowledgments

We thank Rika Nagashima for her technical assistance.

Footnotes

The online-only Data Supplement is available with this article at https://www.ahajournals.org/journal/str/doi/suppl/10.1161/STROKEAHA.118.021467.

Correspondence to Takeo Abumiya, MD, PhD, Department of Neurosurgery, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-Ku, Sapporo, 060-8638, Japan. Email

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